2,3-Butanediol Production, Methyl Ethyl Ketone Production, and Induction of Drought Tolerance in Plants

ABSTRACT

Provided herein are compositions and methods for the fermentative production of 2,3-butanediol (2,3-BDO), compositions and methods for making methyl ethyl ketone (MEK), and methods of inducing drought tolerance in plants.

PRIORITY

This application claims the benefit of U.S. Ser. No. 63/358,527, whichwas filed on Jul. 6, 2022, which is incorporated by reference herein inits entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under contract numberDE-SC0018420 awarded by the Department of Energy. The United StatesGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Aug. 23, 2023, isnamed 743861_SL.xml and is 78,382 bytes in size.

BACKGROUND

Due to global warming and a changing climate, research on the productionof biofuels and chemicals from renewable resources has been steadilyprogressing in recent decades [1, 2]. One such example is the productionof 2,3-butanediol (2,3-BDO), a multi-functional chemical with numerousindustrial applications such as softening agents, plasticizers, drugs,and cosmetics [3, 4]. Intriguingly, a recent study suggested a potentialusage of 2,3-BDO as a Mars-specific rocket propellant [5]. Also, 2,3-BDOcan be used as a platform chemical for producing methyl ethyl ketone(MEK). MEK is a common industrial solvent in paints and coatingformulations [6]. Moreover, MEK can be used as an efficient fueladditive to produce high-quality aviation fuels because it offers ahigher heat of combustion than ethanol [7]. The global MEK market sizeis anticipated to reach USD 4.01 billion and its production to 2.11million tons by 2024 [8]. Currently, MEK is mainly produced using atwo-step chemical process based on the hydration of butylene tosecondary butanol and then dehydration to MEK [9]. However, this processrequires high investment costs and poses serious equipment corrosion andenvironmental issues [10]. As the catalytic dehydration of 2,3-BDO intoMEK is reported with yields up to 95% [3, 11], an integrated conversionprocess consisting of biological 2,3-BDO production and catalyticdehydration of 2,3-BDO would have the potential for greater financialviability and environmental benefits than those of the conventionalchemical process [6].

Most studies on the biological production of 2,3-BDO have been limitedto bacteria such as Klebsiella, Enterobacter, and Bacillus species.However, most of these native 2,3-BDO producers are classified as RiskGroup 2 pathogens (pathogenic to humans), which hinders theirapplicability in industrial 2,3-BDO production [3, 4].

In the past 2,3-BDO titers, yields, and productivities by engineeredyeast such as S. cerevisiae were inferior to bacterial 2,3-EDO producersbecause of two major metabolic limitations. First, ethanol production isa barrier to efficient 2,3-EDO production by S. cerevisiae. To redirectcarbon flux toward 2,3-EDO from ethanol production, pyruvatedecarboxylase (Pdc) or alcohol dehydrogenase (Adh) isozymes have beendeleted in engineered S. cerevisiae strains [12-15]. AlthoughPdc-deficient (Pdc⁻) and Adh-deficient (Adh⁻) strains produced 2,3-EDOas a major product without ethanol production, they exhibited severegrowth defects on glucose medium due to a limited synthesis of cytosolicacetyl-CoA [13, 16] or accumulation of toxic intermediates such asacetaldehyde and acetate [14]. Second, redox imbalance caused byeliminating Pdc and Adh isozymes resulted in impaired cell growth andglycerol accumulation during 2,3-EDO production, leading to low 2,3-EDOproductivities and yields [13, 15, 16]. The chemical properties ofglycerol and 2,3-EDO are similar, so downstream processing forpurification could be complicated, increasing the cost. Previously,partial restoration of Pdc activity [17, 18], redirection of pyruvatecarbon flux into 2,3-EDO biosynthesis [12], introduction of aheterologous NADH oxidase [13, 15, 17-19], and deletion of the glycerolproduction pathway [15, 19] have been attempted to improve 2,3-EDOproduction by engineered S. cerevisiae. Nonetheless, insufficient2,3-EDO productivity and yield as compared to 2,3-EDO titer are still ahurdles for industrial 2,3-EDO production via yeast fermentation [20].

Methods are needed in the art to produce 2,3-BDO.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows a metabolic pathway for 2,3-butanediol (2,3-BDO)production. Two molecules of pyruvate are converted to one molecule of2,3-butanediol via α-acetolactate and acetoin by sequential actions ofα-acetolactate synthase (AlsS), α-acetolactate decarboxylase (AlsD), and2,3-butanediol dehydrogenase (Bdh1). Dashed arrows indicate multipleenzymatic steps. GAP, glycerol-3-phosphate; DHAP, dihydroxyacetonephosphate; G3P, glycerol-3-phophate.

FIG. 2 panels (a)-(d) shows batch fermentation profiles of (a) the CT2strain, (b) the CTL strain, (c) the CTLA strain, and (d) the CTLAPstrain in YPD40 (YP medium with 40 g/L of glucose) under oxygen-limitedconditions. Symbols: OD600 (closed circle), glucose (hexagon), lactate(rectangular), ethanol (open circle), and glycerol (triangle up).Results are the mean of duplicated experiments and error bars representstandard deviations.

FIG. 3 panels (a)-(c) shows a comparison of phenotypic and genotypicchanges between the CTLAB and the CTLABM strains. Batch fermentationprofiles of (a) the CTLAB strain and (b) the CTLABM strain in YPD40under oxygen-limited conditions. (c) The/dhA gene alignments of theCTLAB strain (SEQ ID NO:68) and the CTLABM strain (SEQ ID NO:69). The117^(th) amino acid (valine) of the lactate dehydrogenase (LDH) ismissing in the CTLABM strain. Results are the mean of duplicatedexperiments and error bars represent standard deviations. Fiqurediscloses SEQ ID NOS 68-69 and 78-79, respectively, in order ofappearance.

FIG. 4 panels (a)-(b) shows the effects of deletion of Gpd isozymes on(a) glycerol and ethanol yields (g/g), (b) 2,3-BDO productivities(g/L/h) and yields (g/g) of the engineered yeast strains (CTLABM,CTLABG1, CTLABG2, and CTLABG1G2) in YPD40 under oxygen-limitedconditions. Results are the mean of duplicated experiments and errorbars represent standard deviations.

FIG. 5 panels (a)-(d) shows recovering the redox imbalance from thedeletion of Pdc, Adh, and Gpd isozymes by employing a Pyruvate-Malate(PM) cycle. (a) A schematic diagram of the PM cycle, (b) ethanol yields(g/g), (c) 2,3-BDO productivities (g/L/h), and 2,3-BDO yields (g/gglucose) of the engineered yeast strains (CBMM, CBMMP1, CBMMP2, andCBMMP1P2) in YPD100 under aerobic conditions, and (d) comparison of theintracellular ratios of NADPH to NADP⁺ in the CBMM strain and theCBMMP1P2 strain.

FIG. 6 shows a fed-batch fermentation profile of the CBMMP1P2 strain.Symbols: OD₆₀₀ (closed circle), glucose (hexagon), acetoin (triangledown), ethanol (open circle), glycerol (triangle up), 2,3-BDO (diamond).

FIG. 7 panels (a)-(c) shows uncertainties (box-and-whisker plots) andbreakdowns (stacked bar charts) of (a) minimum product selling price(MPSP), (b) 100-year global warming potential (GWP₁₀₀), and (c) fossilenergy consumption (FEC) per kg of methyl ethyl ketone (MEK) producedvia 2,3-BDO from neutral fermentation of glucose and xylose by S.cerevisiae. Whiskers, boxes, and the middle line represent5^(th)/95^(th) 25^(th)/75^(th), and 50^(th) percentiles, respectively,from 2,000 Monte Carlo simulations. Diamonds and stacked bar chartsreport results for baseline values. For MPSP, the shaded gray regionshows the market price range for MEK ($1.40-1.98/kg). For GWP₁₀₀ andFEC, gray lines marked with lowercase Roman numerals indicate (i.)petroleum-based MEK (GREET 2020) [55] and (ii.) petroleum-based MEK(ecoinvent 3.7.1) [56]. The breakdown method and categories areconsistent with Bhagwat et al. [42]. Tabulated data breaking downcapital and material costs, heating and cooling duties, electricityusage, GWP₁₀₀, and FEC are available online [41].

FIG. 8 panels (a)-(c) shows (a) MPSP, (b) GWPtoo, and (c) FEC of MEKacross a range of 2,3-BDO titer-yield combinations at a productivity of1.0 g/L/h. The triangle indicates the baseline result from the CBMMP1P2strain demonstrated in this study (2,3-BDO titer of 109.9 g/L andoverall yield of 0.334 g/g on glucose and xylose; yield on glucose being0.360 g/g and yield on xylose being 0.288 g/g). The blank area on thetop left indicates high-titer, low-yield combinations that cannot beachieved under current design.

FIG. 9 panels (a)-(e) shows effect of yeast 2,3-BDO fermentation brothon drought tolerance of plants. (a)-(d) Photos of Arabidopsis thalianagrown in a growth chamber. (a) and (b) Plants before treatments anddrought, (c) plants treated with water as a control and (d) plantstreated with 2,3-BDO. (e) The box plots show the median (central line),the lower and upper quartiles (box) and the minimum and maximum values(whiskers). The p-value was calculated using one-way ANOVA (n=3).

FIG. 10 panels (a)-(b) shows a schematic diagram for (a) Pdc-deficientstrain and (b) Pdc1 and Adh1 (pdc1Δ and adh1Δ) deleted strain for2,3-BDO production.

FIG. 11 shows a comparison of the glucose consumption rates of theengineered yeast strains (CTLABM, CTLABG1, CTLABG2, and CTLABG1G2).Results are the mean of duplicated experiments and error bars representstandard deviations.

FIG. 12 shows minimum product selling price (MPSP) of methyl ethylketone (MEK, produced from 2,3-BDO) with uncertainty across overallyield of glycerol on glucose and xylose (x-axis). Dotted lines, dashedlines, and the middle line represent 5^(th)/95^(th), 25^(th)/75^(th) and50^(th) percentiles, respectively, from 500 Monte Carlo simulations ateach of 25 values for overall yield of glycerol on glucose and xylose.The region shaded gray indicates the market price range ($1.40-1.98/kg).

FIG. 13 shows a simplified process flow diagram for (a) conversion(saccharification and fermentation) of pretreated lignocellulosicbiomass into crude 2,3-BDO and (b) separation and upgradation of 2,3-BDOto MEK and IBO. Full process flowsheets are available in the onlinebiorefinery repository [6].

SUMMARY

Provided herein are recombinant yeast cells comprising 1, 2, 3, 4, 5, 6,7, or 8 of the following:

-   -   (a) a genetic modification to reduce or eliminate expression of        glyceraldehyde-3-phosphate dehydrogenase encoded by GPD1 and        GPD2;    -   (b) a genetic modification to reduce or eliminate expression of        pyruvate decarboxylase encoded by PDC1;    -   (c) a genetic modification to reduce or eliminate expression of        alcohol dehydrogenase encoded by ADH1;    -   (d) a heterologous nucleic acid molecule encoding acetolactate        synthase (alsS); (e) a heterologous nucleic acid molecule        encoding acetolactate decarboxylase (alsD);    -   (f) a genetic modification to increase expression of pyruvate        decarboxylase encoded by PYC1 and PYC2 as compared to expression        of pyruvate decarboxylase in a wild-type yeast cell;    -   (g) a heterologous nucleic acid molecule encoding malate        dehydrogenase (Mdh3); and/or    -   (h) a heterologous nucleic acid molecule encoding malic enzyme        (Me1).

A recombinant yeast cell can further comprise 1, 2, 3, 4, or 5 of thefollowing:

-   -   (i) a heterologous nucleic acid molecule encoding xylose        reductase (Xyl1);    -   (j) a heterologous nucleic acid molecule encoding xylitol        dehydrogenase (Xyl2);    -   (k) a heterologous nucleic acid molecule encoding xylulokinase        (Xyl3);    -   (l) a genetic modification to reduce or eliminate expression of        4-nitrophenylphosphatase (Pho13); and/or    -   (m) a genetic modification to reduce or eliminate expression        cytosolic aldehyde dehydrogenase (Ald6).

A recombinant yeast cell can further comprise a heterologous nucleicacid molecule encoding butanediol dehydrogenase (Bdh1).

A heterologous nucleic acid molecule encoding a heterologous nucleicacid molecule encoding acetolactate synthase (alsS) can be derived fromBacillus subtilis.

A heterologous nucleic acid molecule encoding a heterologous nucleicacid molecule encoding acetolactate synthase (alsD) can be derived fromBacillus subtilis.

A genetic modification to increase expression of pyruvate decarboxylaseencoded by PYC1 and PYC2 can comprise a strong promoter operably linkedto the PYC1 and PYC2. A strong promoter can be a TEF1 promoter, a PGK1promoter, or any other suitable promoter.

A heterologous nucleic acid molecule encoding malate dehydrogenase(Mdh3) can be derived from Saccharomyces cerevisiae.

A nucleic acid molecule encoding Mdh3 can encode a truncated Mdh3(tMdh3), wherein the last three amino acids (SKL) are absent. A nucleicacid molecule encoding Mdh3 can be operably linked to a strong promoter.

A heterologous nucleic acid molecule encoding malic enzyme (Me1) can bederived from Rhodosporidium toruloides.

A heterologous nucleic acid molecule encoding xylose reductase (Xyl1),xylitol dehydrogenase (Xyl2), and xylulokinase (Xyl3) can be derivedfrom Scheffersomyces stipitis.

A recombinant yeast cell can be Saccharomyces. The recombinant yeastcell can be capable of fermenting xylose.

An aspect provides a yeast cell culture comprising two or more of therecombinant yeast cells described herein.

Another aspect provides a method of producing 2,3-butanediol (2,3-BDO)comprising contacting a substrate with any of the recombinant yeastcells described herein. The substrate can be lignocellulosic orcellulosic feedstock. The substrate can comprise glycose, xylose, or acombination of glucose and xylose. Substantially no glycerol or ethanolcan be accumulated in the fermentation broth or fermentation media. Lessthan 2 g/L of ethanol and less than 2 g/L of glycerol can be accumulatedin the fermentation broth or fermentation media. 2,3-BDO can be producedat more than 0.5 g/L/h or at more than 1.0 g/L/h. 2,3-BDO can beproduced at a yield of 100 g/L or more.

Yet another aspect provides a method of producing methyl ethyl ketone(MEK). The method can comprise:

-   -   (a) contacting a substrate with any recombinant yeast cell        described herein under fermentation conditions;    -   (b) collecting and purifying 2,3-BDO to form purified 2,3-BDO;    -   (c) subjecting the purified 2,3-BDO to catalytic dehydration        such that MEK is produced. A catalyst for the catalytic        dehydration can be tricalcium phosphate or other suitable        catalyst. The purified 2,3-BDO can be greater than 90 wt % pure.

Even another aspect provides a fermentation broth produced by contactingany recombinant yeast cell described herein with a fermentation medium.

An aspect provides a method of inducing drought tolerance in plantscomprising contacting roots of the plants with a fermentation brothproduced by contacting any recombinant yeast cell described herein witha fermentation medium. The survival rate of the plants can be at least2-fold higher than plants not contacted with the fermentation broth. Thefermentation broth can contain substantially no ethanol. Thefermentation broth can contain substantially no glycerol. Thefermentation broth can comprise 200 μM or more of 2,3-BDO.

Rising concerns for sustainability and global climate change have driventhe development of sustainable production pathways for biofuels andchemicals from lignocellulosic biomass via integrated biological andchemical processes. Therefore, provided herein are engineered yeastcapable of producing 2,3-butanediol (2,3-BDO) from lignocellulosichydrolysates, cellulosic hydrolysates, and other suitable substrateswith a high yield and productivity. Also provided are methods ofproduction of methyl ethyl ketone (MEK) through catalytic dehydration of2,3-BDO. Engineered yeast can produce 2,3-BDO without accumulatingglycerol and/or ethanol, which hinders downstream processing of 2,3-BDO.Furthermore, fermentation broth containing 2,3-BDO can be used as abiostimulant inducing drought tolerance in plants.

DETAILED DESCRIPTION

To circumvent limitations of previous attempts to produce 2,3-BDO withrecombinant bacteria and yeast, engineered yeast strains were produced,which are capable of producing 2,3-BDO with a high productivity andwithout by-product production through extensive metabolic reprogramming(FIG. 1 ). First, we deleted major the isozymes of Pdc and Adh (pdc1Δand adh1Δ) to minimize ethanol production while maintaining sufficientlevels of acetyl-CoA for cell growth (FIG. 10 ). Second, an alsS genecoding for acetolactate synthase and alsD gene coding for acetolactatedecarboxylase were overexpressed to enhance metabolic fluxes toward2,3-BDO biosynthesis. Third, Gpd isozymes (gpd1Δ and gpd2Δ) were deletedto eliminate glycerol production. Fourth, we introduced an NAD⁺regenerating Pyruvate-Malate (PM) cycle consisting of endogenous PYC1and PYC2 genes coding for pyruvate carboxylase, a truncated MDH3 (tMDH3)gene coding for malate dehydrogenase and ME1 gene coding for malicenzyme, to restore the redox imbalance caused by the elimination ofglycerol production. Lastly, the expression levels of PYC1 and PYC2genes were increased to improve the NAD⁺ regenerating capability of thePM cycle. As a result, our engineered strains produced 2,3-BDO with ahigh productivity without ethanol or glycerol production, which pavesthe way toward implementation and financially viable downstreamprocessing of 2,3-BDO.

2,3-BDO also has various benefits for plants, such as growth promotion,heat resistance, and disease resistance. Drought is one of the majorthreats to crop production worldwide. In the U.S., nearly 67% of croplosses reported in the last 50 years were due to drought stress.Intriguingly, evidence from many studies suggests that 2,3-BDO andacetoin trigger hormonal responses inducing systemic drought tolerancein plants. Most studies have focused on understanding the physiologicalrole of 2,3-BDO and acetoin in plants under drought stress by eitherroot colonization with rhizobacteria or root treatment of pure 2,3-BDOand acetoin chemicals. However, the 2,3-BDO delivery methods cannot beapplied as a practical solution for field and industrial crops undersevere drought stress due to economic and sustainability concerns. Tosolve this, we established a simple and effective strategy for inducingdrought tolerance in plants using yeast 2,3-BDO fermentation broth as abiostimulant without purification processes.

Additionally, we designed, simulated, and evaluated biorefineriesproducing MEK from lignocellulosic hydrolysates via yeast 2,3-BDOfermentation and catalytic dehydration of 2,3-BDO using BioSTEAM [23,24]—an open-source platform—as a tool. To efficiently produce MEK fromlignocellulosic biomass, we devised an integrated conversion processcomprising (i) a biological process using a metabolically engineeredyeast producing high levels of 2,3-BDO without ethanol and glycerolproduction and (ii) a chemical process that dehydrates 2,3-BDO to MEKusing a catalyst. We evaluated the financial viability and environmentalbenefits of MEK-producing biorefineries through design, simulation,techno-economic analysis (TEA), and life cycle assessment (LCA) underuncertainty. This study demonstrated the feasibility of cost-competitiveand sustainable bio-based MEK production via yeast 2,3-BDO fermentationfrom lignocellulosic biomass.

Polynucleotides and Genes

Polynucleotides contain less than an entire microbial genome and can besingle- or double-stranded nucleic acids. A polynucleotide can be RNA,DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA orcombinations thereof. A polynucleotide can comprise, for example, agene, open reading frame, non-coding region, or regulatory element.

A gene is any polynucleotide molecule that encodes a polypeptide,protein, or fragment thereof, optionally including one or moreregulatory elements preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. In one embodiment, a genedoes not include regulatory elements preceding and following the codingsequence. A native or wild-type gene refers to a gene as found innature, optionally with its own regulatory elements preceding andfollowing the coding sequence. A chimeric or recombinant gene refers toany gene that is not a native or wild-type gene, optionally comprisingregulatory elements preceding and following the coding sequence, whereinthe coding sequences and/or the regulatory elements, in whole or inpart, are not found together in nature. Thus, a chimeric gene orrecombinant gene can comprise regulatory elements and coding sequencesthat are derived from different sources, or regulatory elements andcoding sequences that are derived from the same source, but arrangeddifferently than is found in nature. A gene can encompass full-lengthgene sequences (e.g., as found in nature and/or a gene sequence encodinga full-length polypeptide or protein) and can also encompass partialgene sequences (e.g., a fragment of the gene sequence found in natureand/or a gene sequence encoding a protein or fragment of a polypeptideor protein). A gene can include modified gene sequences (e.g., modifiedas compared to the sequence found in nature). Thus, a gene is notlimited to the natural or full-length gene sequence found in nature.

Polynucleotides can be purified free of other components, such asproteins, lipids and other polynucleotides. For example, thepolynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%purified. A polynucleotide existing among hundreds to millions of otherpolynucleotide molecules within, for example, cDNA or genomic libraries,or gel slices containing a genomic DNA restriction digest are not to beconsidered a purified polynucleotide. Polynucleotides can encode thepolypeptides described herein (e.g., an acetolactate synthase (AlsS)polypeptide, an acetolactate decarboxylase (AlsD) polypeptide, a malatedehydrogenase (Mdh3) polypeptide, a truncated malate dehydrogenase(tMdh3) polypeptide, a malic enzyme (Me1) polypeptide, a xylosereductase (Xyl1) polypeptide, a xylitol dehydrogenase (Xyl2)polypeptide, a xylulokinase (Xyl3) polypeptide, a (R,R)-butanedioldehydrogenase (Bdh1) polypeptide, a glyceraldehyde-3-phosphatedehydrogenase (Gpd1) polypeptide, a glyceraldehyde-3-phosphatedehydrogenase (Gpd2) polypeptide, a pyruvate decarboxylase (Pdc1)polypeptide, an alcohol dehydrogenase (Adh1) polypeptide, a4-nitrophenylphosphatase (Pho13) polypeptide, a cytosolic aldehydedehydrogenase (Ald6) polypeptide, a pyruvate decarboxylase (Pyc1)polypeptide, a pyruvate decarboxylase (Pyc2) polypeptide) and mutants orvariants thereof.

Polynucleotides can comprise other nucleotide sequences, such assequences coding for linkers, signal sequences, TMR stop transfersequences, transmembrane domains, or ligands useful in proteinpurification such as glutathione-S-transferase, histidine tag, andStaphylococcal protein A.

Polynucleotides can be codon optimized for expression in yeast, such asSaccharomyces, e.g., Saccharomyces cerevisiae.

Polynucleotides can be isolated. An isolated polynucleotide is anaturally-occurring polynucleotide that is not immediately contiguouswith one or both of the 5′ and 3′ flanking genomic sequences that it isnaturally associated with. An isolated polynucleotide can be, forexample, a recombinant DNA molecule of any length, provided that thenucleic acid sequences naturally found immediately flanking therecombinant DNA molecule in a naturally-occurring genome is removed orabsent. Isolated polynucleotides also include non-naturally occurringnucleic acid molecules. Polynucleotides can encode full-lengthpolypeptides, polypeptide fragments, and variant or fusion polypeptides.

Degenerate polynucleotide sequences encoding polypeptides describedherein, as well as homologous nucleotide sequences that are at leastabout 80, or about 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to polynucleotides described herein and the complementsthereof are also polynucleotides. Degenerate nucleotide sequences arepolynucleotides that encode a polypeptide described herein or fragmentsthereof, but differ in nucleic acid sequence from the wild-typepolynucleotide sequence, due to the degeneracy of the genetic code.Complementary DNA (cDNA) molecules, species homologs, and variants ofpolynucleotides that encode biologically functional polypeptides alsoare polynucleotides.

Polynucleotides can be obtained from nucleic acid sequences present in,for example, a microorganism such as a yeast or bacterium.Polynucleotides can also be synthesized in the laboratory, for example,using an automatic synthesizer. An amplification method such as PCR canbe used to amplify polynucleotides from either genomic DNA or cDNAencoding the polypeptides.

Polynucleotides can be, for example, acetolactate synthase (alsS),acetolactate decarboxylase (alsD), malate dehydrogenase (mdh3) (tmdh3),malic enzyme (ME1), xylose reductase (XYL1), xylitol dehydrogenase(XYL2), xylulokinase (XYL3), (R,R)-butanediol dehydrogenase (BDH1),glyceraldehyde-3-phosphate dehydrogenase (GPD1),glyceraldehyde-3-phosphate dehydrogenase (GPD2), pyruvate decarboxylase(PDC1), alcohol dehydrogenase (ADH1), 4-nitrophenylphosphatase (PHO13),cytosolic aldehyde dehydrogenase (ALD6), pyruvate decarboxylase (PYC1),pyruvate decarboxylase (PYC2), malate dehydrogenase (mdh3), truncatedmalate dehydrogenase (tmdh3), pyruvate decarboxylase (PYC1), andpyruvate decarboxylase (PYC2).

Polynucleotides can comprise coding sequences for naturally occurringpolypeptides or can encode altered sequences that do not occur innature.

Unless otherwise indicated, the term polynucleotide or gene includesreference to the specified sequence as well as the complementarysequence thereof. The expression products of genes or polynucleotidesare often proteins, or polypeptides, but in non-protein coding genessuch as rRNA genes or tRNA genes, the product is a functional RNA. Theprocess of gene expression is used by all known life forms, i.e.,eukaryotes (including multicellular organisms), prokaryotes (bacteriaand archaea), and viruses, to generate the macromolecular machinery forlife. Several steps in the gene expression process can be modulated,including the transcription, up-regulation, RNA splicing, translation,and post-translational modification of a protein.

Any process that deletes, reduces, or attenuates the expression of e.g.,a Gpd1 polypeptide, a Gpd2 polypeptide, a Pdc1 polypeptide, an Adh1polypeptide, a Pho13 polypeptide, or an Ald6 polypeptide can be used tomake a microorganism described herein. Any process that adds orincreases the expression of e.g., an AlsS polypeptide, an AlsDpolypeptide, a Mdh3 polypeptide, a tMdh3 polypeptide, a Me1 polypeptide,a Bdh1 polypeptide, a Xyl1 polypeptide, a Xyl2 polypeptide, a Xyl3polypeptide, a Pyc1 polypeptide, Pyc2 polypeptide, (R,R)-butanedioldehydrogenase (Bdh1) can be used to make a microorganism describedherein.

Polypeptides

A polypeptide is a polymer of two or more amino acids covalently linkedby amide bonds. A polypeptide can be post-translationally modified. Apurified polypeptide is a polypeptide preparation that is substantiallyfree of cellular material, other types of polypeptides, chemicalprecursors, chemicals used in synthesis of the polypeptide, orcombinations thereof. A polypeptide preparation that is substantiallyfree of cellular material, culture medium, chemical precursors,chemicals used in synthesis of the polypeptide, etc., has less thanabout 30%, 20%, 10%, 5%, 1% or more of other polypeptides, culturemedium, chemical precursors, and/or other chemicals used in synthesis.Therefore, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% ormore pure. A purified polypeptide does not include unpurified orsemi-purified cell extracts or mixtures of polypeptides that are lessthan 70% pure.

The term “polypeptides” can refer to one or more of one type ofpolypeptide (a set of polypeptides). “Polypeptides” can also refer tomixtures of two or more different types of polypeptides (a mixture ofpolypeptides). The terms “polypeptides” or “polypeptide” can each alsomean “one or more polypeptides.” As used herein, the term “polypeptideof interest” or “polypeptides of interest”, “protein of interest”,“proteins of interest” includes any or a plurality of any of, e.g.,AlsS, AlsD, Mdh3, tMdh3, Me1, Bdh1, Xyl1, Xyl2, Xyl3, Gpd1, Gpd2, Pdc1,Adh1, Pho13, Ald6, Pyc1, Bhd1, Pyc2 and/or Pyc2 polypeptides or otherpolypeptides described herein.

A mutated protein or polypeptide comprises at least one deleted,inserted, and/or substituted amino acid, which can be accomplished viamutagenesis of polynucleotides encoding these amino acids. Mutagenesisincludes well-known methods in the art, and includes, for example,site-directed mutagenesis by means of PCR or viaoligonucleotide-mediated mutagenesis as described in Sambrook et al.,Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989).

As used herein, the term “sufficiently similar” means a first amino acidsequence that contains a sufficient or minimum number of identical orequivalent amino acid residues relative to a second amino acid sequencesuch that the first and second amino acid sequences have a commonstructural domain and/or common functional activity. For example, aminoacid sequences that comprise a common structural domain that is at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or at least about 100%, identical aredefined herein as sufficiently similar will be sufficiently similar tothe amino acid sequence of the polypeptides described herein. Suchvariants generally retain the functional activity of the polypeptidesdescribed herein. Variants include peptides that differ in amino acidsequence from the native and wild-type peptide, respectively, by way ofone or more amino acid deletion(s), addition(s), and/or substitution(s).These may be naturally occurring variants as well as artificiallydesigned ones.

The terms “sequence identity” or “percent identity” are usedinterchangeably herein. To determine the percent identity of twopolypeptide molecules or two polynucleotide sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe sequence of a first polypeptide or polynucleotide for optimalalignment with a second polypeptide or polynucleotide sequence). Theamino acids or nucleotides at corresponding amino acid or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid or nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=number of identical positions/total number ofpositions (i.e., overlapping positions)×100). In some embodiments thelength of a reference sequence aligned for comparison purposes is atleast 80% of the length of the comparison sequence, and in someembodiments is at least 90% or 100%. In an embodiment, the two sequencesare the same length.

Ranges of desired degrees of sequence identity are approximately 70% to100% and integer values in between. Percent identities between adisclosed sequence and a claimed sequence can be at least 70%, at least80%, at least 83%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, at least 99.5%, or at least 99.9%. In general, anexact match indicates 100% identity over the length of the referencesequence. Polypeptides and polynucleotides that are sufficiently similarto polypeptides (e.g., AIsS, AIsD, Mdh3, tMdh3, Me1, Bdh1, Xyl1, Xyl2,Xyl3, Gpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6, Pyc1, Pyc2) andpolynucleotides described herein (e.g., alsS, alsD, BDH1, mdh3, tmdh3,ME1, XYL1, XYL2, XYL3, GPD1, GPD2, PDC1, ADH1, PHO13, ALD6, PYC1, PYC2polynucleotides) can be used herein. Polypeptides and polynucleotidesthat are about 70, 80, 85, 90, 91, 92, 93, 94 95, 96, 97, 98, 99 99.5%or more identical to polypeptides and polynucleotides described hereincan also be used herein.

Polypeptides and polynucleotides that are about 70, 80, 85, 90, 95, 96,97, 98, 99% or more identity to polypeptides (e.g., AIsS, AIsD, Mdh3,tMdh3, Me1, Bdh1, Xyl1, Xyl2, Xyl3, Gpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6,Pyc1, Pyc2) and polynucleotides (e.g., alsS, alsD, mdh3, tmdh3, ME1,BDH1, XYL1, XYL2, XYL3, GPD1, GPD2, PDC1, ADH1, PHO13, ALD6, PYC1, PYC2polynucleotides) described herein can also be used.

Constructs and Cassettes

A recombinant construct is a polynucleotide having heterologouspolynucleotide elements. Recombinant constructs include expressioncassettes or expression constructs, which refer to an assembly that iscapable of directing the expression of a polynucleotide or gene ofinterest. An expression cassette generally includes regulatory elementssuch as a promoter that is operably linked to (so as to directtranscription of) a polynucleotide and often includes a polyadenylationsequence as well.

An “expression cassette” refers to a fragment of DNA comprising a codingsequence of a selected polynucleotide or gene (e.g., alsS, alsD, mdh3,tmdh3, ME1, BDH1, XYL1, XYL2, XYL3, GPD1, GPD2, PDC1, ADH1, PHO13, ALD6,PYC1, and/or PYC2 polynucleotides) and, optionally, regulatory elementspreceding (5′ non-coding sequences) and following (3′ non-codingsequences) the coding sequence can be required for expression of theselected gene product. Thus, an expression cassette is typicallycomposed of: 1) a promoter sequence; 2) a coding sequence (“ORF”); andoptionally 3) a 3′ untranslated region (i.e., a terminator). Theexpression cassette is usually included within a vector, to facilitatecloning and transformation. Different expression cassettes can betransformed into different organisms including bacteria, yeast, plants,and mammalian cells, as long as the correct regulatory elements are usedfor each host.

A recombinant construct or expression cassette can be contained within avector. In addition to the components of the recombinant construct, thevector can include, one or more selectable markers, a signal whichallows the vector to exist as single-stranded DNA (e.g., a M13 origin ofreplication), at least one multiple cloning site, and an origin ofreplication (e.g., a SV40 or adenovirus origin of replication).

Generally, a polynucleotide or gene that is introduced into agenetically engineered organism is part of a recombinant construct. Apolynucleotide can comprise a gene of interest, e.g., a coding sequencefor a protein, or can be a sequence that is capable of regulatingexpression of a gene, such as a regulatory element like a promoter, anantisense sequence, a sense suppression sequence, or a miRNA sequence. Arecombinant construct can include, for example, regulatory elementsoperably linked 5′ or 3′ to a polynucleotide encoding one or morepolypeptides of interest. For example, a promoter can be operably linkedwith a polynucleotide encoding one or more polypeptides of interest whenit is capable of affecting the expression of the polynucleotide (i.e.,the polynucleotide is under the transcriptional control of thepromoter). Polynucleotides can be operably linked to regulatory elementsin sense or antisense orientation. The expression cassettes orrecombinant constructs can additionally contain a 5′ leaderpolynucleotide. A leader polynucleotide can contain a promoter as wellas an upstream region of a gene. The regulatory elements (i.e.,promoters, enhancers, transcriptional regulatory regions, translationalregulatory regions, and translational termination regions) and/or thepolynucleotide encoding a signal anchor can be native/analogous to thehost cell or to each other. Alternatively, the regulatory elements canbe heterologous to the host cell or to each other. See, U.S. Pat. No.7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and2006/0248616. The expression cassette or recombinant construct canadditionally contain one or more selectable marker genes.

Methods for preparing polynucleotides operably linked to regulatoryelements and expressing polypeptides in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide can beoperably linked when it is positioned adjacent to or close to one ormore regulatory elements, which direct transcription and/or translationof the polynucleotide.

A promoter is a nucleotide sequence that is capable of controlling theexpression of a coding sequence or gene. Promoters are generally located5′ of the sequence that they regulate. Promoters may be derived in theirentirety from a native gene, can be composed of different elementsderived from promoters found in nature, and/or comprise syntheticnucleotide segments. Those skilled in the art will readily ascertainthat different promoters may regulate expression of a coding sequence orgene in response to a particular stimulus, e.g., in a cell- ortissue-specific manner, in response to different environmental orphysiological conditions, or in response to specific compounds.Promoters are typically classified into two classes: inducible andconstitutive. A constitutive promoter refers to a promoter that allowsfor continual transcription of the coding sequence or gene under itscontrol.

An inducible promoter refers to a promoter that initiates increasedlevels of transcription of the coding sequence or gene under its controlin response to a stimulus or an exogenous environmental condition. Ifinducible, there are inducer polynucleotides present therein thatmediate regulation of expression so that the associated polynucleotideis transcribed only when an inducer molecule is present. A directlyinducible promoter refers to a regulatory region, wherein the regulatoryregion is operably linked to a gene encoding a protein or polypeptide,where, in the presence of an inducer of the regulatory region, theprotein or polypeptide is expressed. An indirectly inducible promoterrefers to a regulatory system comprising two or more regulatory regions,for example, a first regulatory region that is operably linked to afirst gene encoding a first protein, polypeptide, or factor, e.g., atranscriptional regulator, which is capable of regulating a secondregulatory region that is operably linked to a second gene, the secondregulatory region may be activated or repressed, thereby activating orrepressing expression of the second gene. Both a directly induciblepromoter and an indirectly inducible promoter are encompassed byinducible promoter.

A promoter can be any polynucleotide that shows transcriptional activityin the chosen host microorganism. A promoter can be naturally-occurring,can be composed of portions of various naturally-occurring promoters, ormay be partially or totally synthetic. Guidance for the design ofpromoters is derived from studies of promoter structure, such as that ofHarley and Reynolds, Nucleic Acids Res., 15, 2343-61 (1987). Inaddition, the location of the promoter relative to the transcriptionstart can be optimized. Many suitable promoters for use inmicroorganisms and yeast are well known in the art, as arepolynucleotides that enhance expression of an associated expressiblepolynucleotide.

A selectable marker can provide a means to identify microorganisms thatexpress a desired product. Selectable markers include, but are notlimited to, ampicillin resistance for prokaryotes such as E. coli,neomycin phosphotransferase, which confers resistance to theaminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella,EMBO J. 2:987-995, (1983)); dihydrofolate reductase, which confersresistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.)13:143-149, (1994)); trpB, which allows cells to utilize indole in placeof tryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, (1988));mannose-6-phosphate isomerase which allows cells to utilize mannose (WO94/20627); hygro, which confers resistance to hygromycin (Marsh, Gene32:481-485, (1984)); ornithine decarboxylase, which confers resistanceto the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine (DFMO; McConlogue, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.,(1987)); deaminase from Aspergillus terreus, which confers resistance toBlasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59:2336-2338,(1995)); phosphinothricin acetyltransferase gene, which confersresistance to phosphinothricin (White et al., Nucl. Acids Res. 18:1062,(1990); Spencer et al., Theor. Appl. Genet. 79:625-633, (1990)); amutant acetolactate synthase, which confers imidazolione or sulfonylurearesistance (Lee et al., EMBO J. 7:1241-1248, (1988)), a mutantEPSPV-synthase, which confers glyphosate resistance (Hinchee et al.,BioTechnology 91:915-922, (1998)); a mutant psbA, which confersresistance to atrazine (Smeda et al., Plant Physiol. 103:911-917,(1993)), a mutant protoporphyrinogen oxidase (see U.S. Pat. No.5,767,373), or other markers conferring resistance to an herbicide suchas glufosinate.

A transcription termination region of a recombinant construct orexpression cassette is a downstream regulatory region including a stopcodon and optionally a transcription terminator sequence. Transcriptiontermination regions that can be used can be homologous to thetranscriptional initiation region, can be homologous to thepolynucleotide encoding a polypeptide of interest, or can beheterologous (i.e., derived from another source). A transcriptiontermination region or can be naturally occurring, or wholly or partiallysynthetic. 3′ non-coding sequences encoding transcription terminationregions may be provided in a recombinant construct or expressionconstruct and may be from the 3′ region of the gene from which theinitiation region was obtained or from a different gene. A large numberof termination regions are known and function satisfactorily in avariety of hosts when utilized in both the same and different genera andspecies from which they were derived. Termination regions may also bederived from various genes native to the preferred hosts. Thetermination region is usually selected more for convenience rather thanfor any particular property.

The procedures described herein employ, unless otherwise indicated,conventional techniques of chemistry, molecular biology, microbiology,recombinant DNA, genetics, immunology, cell biology, cell culture andtransgenic biology, which are within the skill of the art. (See, e.g.,Maniatis, et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982); Sambrook, et al., (1989);Sambrook and Russell, Molecular Cloning, 3rd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel, et al.,Current Protocols in Molecular Biology, John Wiley & Sons (includingperiodic updates) (1992); Glover, DNA Cloning, IRL Press, Oxford (1985);Russell, Molecular biology of plants: a laboratory course manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); Anand,Techniques for the Analysis of Complex Genomes, Academic Press, N Y(1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology,Academic Press, N Y (1991); Harlow and Lane, Antibodies, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1988); Nucleic AcidHybridization, B. D. Hames & S. J. Higgins eds. (1984); TranscriptionAnd Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture OfAnimal Cells, R. I. Freshney, A. R. Liss, Inc. (1987); Immobilized CellsAnd Enzymes, IRL Press (1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology, Academic Press,Inc., NY); Methods In Enzymology, Vols. 154 and 155, Wu, et al., eds.;Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker,eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds. (1986);Riott, Essential Immunology, 6th Edition, Blackwell ScientificPublications, Oxford (1988); Fire, et al., RNA Interference TechnologyFrom Basic Science to Drug Development, Cambridge University Press,Cambridge (2005); Schepers, RNA Interference in Practice, Wiley-VCH(2005); Engelke, RNA Interference (RNAi): The Nuts &Bolts of siRNATechnology, DNA Press (2003); Gott, RNA Interference, Editing, andModification: Methods and Protocols (Methods in Molecular Biology),Human Press, Totowa, N.J. (2004); and Sohail, Gene Silencing by RNAInterference: Technology and Application, CRC (2004)).

Recombinant Yeast

A recombinant, transgenic, or genetically engineered yeast has beengenetically modified from its native state. Thus, “recombinant yeast” or“recombinant yeast cell” refers to a yeast cell that has beengenetically modified from the native state. A recombinant yeast cell canhave, for example, nucleotide insertions, nucleotide deletions,nucleotide rearrangements, gene disruptions, recombinantpolynucleotides, heterologous polynucleotides, deleted polynucleotides,nucleotide modifications, or combinations thereof introduced into itsDNA. These genetic modifications can be present in the chromosome of theyeast, or on a plasmid in the yeast. Recombinant yeast cells disclosedherein can comprise exogenous polynucleotides on plasmids.Alternatively, recombinant cells can comprise exogenous polynucleotidesstably incorporated into their chromosome.

A heterologous or exogenous polypeptide or polynucleotide refers to anypolynucleotide or polypeptide that does not naturally occur or that isnot present in the starting target yeast. For example, a polynucleotidefrom bacteria or yeast that is transformed into a yeast cell that doesnaturally or otherwise comprise the yeast polynucleotide is aheterologous or exogenous yeast. A heterologous or exogenous polypeptideor polynucleotide can be a wild-type, synthetic, or mutated polypeptideor polynucleotide. In an embodiment, a heterologous or exogenouspolypeptide or polynucleotide is not naturally present in a startingtarget yeast and is from a different genus or species than the startingtarget yeast. In an aspect, a heterologous or exogenous polypeptide orpolynucleotide is not naturally present in a starting target yeast andis from a bacterium.

A homologous or endogenous polypeptide or polynucleotide refers to anypolynucleotide or polypeptide that naturally occurs or that is otherwisepresent in a starting target yeast. For example, a polynucleotide thatis naturally present in a yeast is a homologous or endogenouspolynucleotide. In an embodiment, a homologous or endogenous polypeptideor polynucleotide is naturally present in a starting target yeast.

A recombinant yeast can comprise one or more polynucleotides not presentin a corresponding wild-type cell, wherein the polynucleotides have beenintroduced into that yeast using recombinant DNA techniques, or whichpolynucleotides are not present in a wild-type yeast and is the resultof one or more mutations.

In an embodiment a genetically engineered or recombinant yeast comprisesone or more heterologous or exogenous polynucleotides, optionallyoperably linked to one or more heterologous, exogenous, or endogenousregulatory elements such that one or more heterologous or exogenousbiologically active polypeptides are expressed by the yeast.

The term “overexpression” or “overexpressed” as used herein refers to alevel of enzyme or polypeptide expression that is greater than what ismeasured in a wild-type cell of the same species as the host cell thathas not been genetically altered. The overexpression of the enzymes orpolypeptides can be achieved by constructing inducible overexpressionvectors encoding for the desired polypeptide. Strong promoters andstrong constitutive promoters can be used to induce overexpression of apolypeptide as can the use of multiple copies of a polynucleotide in therecombinant microorganism. Overexpression is any expression level thatis greater than wild-type expression. In an embodiment a Pyc1polypeptide, a Pyc2 polypeptide, a Mdh3 polypeptide, a tMdh3polypeptide, or combinations thereof can be overexpressed.

Yeast In aspects a yeast of the genus Saccharomyces, e.g., Saccharomycescerevisiae, is genetically engineered using any suitable technique. Insome aspects one or more of alsS, alsD, mdh3, tmdh3, ME1, BDH1, XYL1,XYL2, XYL3, GPD1, GPD2, PDC1, ADH1, PHO13, ALD6, PYC1, PYC2polynucleotides are deleted, rendered non-functional, overexpressed, oradded to a yeast, such as Saccharomyces, e.g., Saccharomyces cerevisiae.Other Saccharomyces species can be used such as S. castellii, S.mikatae, S. cariocanus, S. boulardii, S. paracoxus, and S. kudriavzevii.Other yeast that can be use include Scheffersomyces sp., e.g., S.stipitis, Yarrowia sp. such as Y. lipolytica, Kluyveromyces sp. such asK. lactis, Dekkera sp. such as D. bruxellensis, Candida sp., Kloeckerasp., Hanseniaspora sp., Brettanomyces sp., Pichia sp., and Lanchacea sp.

In an aspect, a recombinant yeast, Saccharomyces, e.g., Saccharomycescerevisiae, comprises a deleted or non-functional GPD1 gene orpolynucleotide. In an aspect, a recombinant yeast, Saccharomyces, e.g.,Saccharomyces cerevisiae, comprises a deleted or non-functional GPD2gene or polynucleotide. In an aspect, a recombinant yeast,Saccharomyces, e.g., Saccharomyces cerevisiae, comprises a deleted ornon-functional PDC1 gene or polynucleotide. In an aspect, a recombinantyeast, Saccharomyces, e.g., Saccharomyces cerevisiae, comprises adeleted or non-functional ADH1 gene or polynucleotide. In an aspect, arecombinant yeast, Saccharomyces, e.g., Saccharomyces cerevisiae,comprises a deleted or non-functional PHO13 gene or polynucleotide. Inan aspect, a recombinant yeast, Saccharomyces, e.g., Saccharomycescerevisiae, comprises a deleted or non-functional ADH6 gene orpolynucleotide.

In an aspect, a recombinant yeast, Saccharomyces, e.g., Saccharomycescerevisiae, comprises a deleted or non-functional GPD1, GPD2, PDC1, andADH1 gene or polynucleotide. Additionally, the recombinant yeast canadditionally lack a PHO13, and/or ALD6 gene or polynucleotide.Alternatively, the recombinant yeast comprises a deleted ornon-functional PHO13, and/or ALD6 gene or polynucleotide.

In an aspect, a recombinant yeast can comprise a recombinant yeast,e.g., Saccharomyces such as S. cerevisiae, comprising a recombinantnucleic acid molecule encoding AIsS. In an aspect, a recombinant yeastcan comprise a recombinant yeast, e.g., Saccharomyces such as S.cerevisiae, comprising a recombinant nucleic acid molecule encodingAIsD. In an aspect, a recombinant yeast can comprise a recombinantyeast, e.g., Saccharomyces such as S. cerevisiae, comprising arecombinant nucleic acid molecule encoding Mdh3. In an aspect, arecombinant yeast can comprise a recombinant yeast, e.g., Saccharomycessuch as S. cerevisiae, comprising a recombinant nucleic acid moleculeencoding tMdh3. In an aspect, a recombinant yeast can comprise arecombinant yeast, e.g., Saccharomyces such as S. cerevisiae, comprisinga recombinant nucleic acid molecule encoding Me1. In an aspect, arecombinant yeast can comprise a recombinant yeast, e.g., Saccharomycessuch as S. cerevisiae, comprising a recombinant nucleic acid moleculeencoding AIsS, AIsD, Mdh3 (and/or tMdh3), and Me1. In an aspect, arecombinant yeast can additionally comprise a recombinant nucleic acidmolecule encoding Xyl1, Xyl2, and Xyl3. A recombinant yeast canadditionally comprise a recombinant nucleic acid molecule encoding Bdh1.

In an aspect, a yeast culture can comprise a recombinant yeast, e.g.,Saccharomyces such as S. cerevisiae, comprising recombinant nucleic acidmolecules encoding alsS, alsD, mdh3, tmdh3, ME1, XYL1, XYL2, XYL3, BDH1.In an aspect, PYC1, PYC2, mdh3, tmdh3, BDH1 polynucleotides or genes areoverexpressed.

Acetolactate Synthase (alsS) and Acetolactate Decarboxylase (alsD)

One or more acetolactate synthase (alsS) and acetolactate decarboxylase(alsD) polynucleotides can be genetically engineered into a targetyeast, e.g., Saccharomyces, so that the yeast can express acetolactatesynthase (AIsS) and acetolactate decarboxylase (AlsD). In an aspect, thegenes encoding the proteins can be obtained from Bacillus, e.g., B.subtilis or other suitable organism.

An alsS gene can encode a polypeptide as shown in UniProtQ04789:

SEQ ID NO: 70MTKATKEQKS LVKNRGAELV VDCLVEQGVT HVFGIPGAKI DAVEDALQDK GPEIIVARHE QNAAFMAQAV GRLTGKPGVV LVTSGPGASN LATGLLTANT EGDPVVALAG NVIRADRLKR THQSLDNAAL FQPITKYSVE VQDVKNIPEA VINAFRIASA GQAGAAFVSF PQDVVNEVIN TKNVRAVAAP KLGPAADDAI SAAIAKIQTA KLPVVLVGMK GGRPEAIKAV RKLLKKVQLP FVETYQAAGT LSRDLEDQYF GRIGLERNQP GDLLLEQADV VLTIGYDPIE YDPKFWNING DRTIIHLDEI IADIDHAYQP DLELIGDIPS TINHIEHDAV KVEFAEREQK ILSDLKQYMH EGEQVPADWK SDRAHPLEIV KELRNAVDDH VTVTCDIGSH AIWMSRYFRS YEPLTLMISN GMQTLGVALP WAIGASLVKP GEKVVSVSGD GGFLESAMEL ETAVRLKAPI VHIVWNDSTY DMVAFQQLKK YNRTSAVDFG NIDIVKYAES FGATGLRVES PDQLADVLRQ GMNAEGPVII DVPVDYSDNI NLASDKLPKE FGELMKTKAL 

In an aspect an alsS polynucleotide can encode a polypeptide having 70,75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQ IDNO:70. In other aspects an alsS gene can encode AlsS polypeptidescomprising 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequenceidentity to AlsS polypeptides from B. subtilis (e.g., GenBank Accessionnumbers WP_251188357, WP_251188357.1, WP_047183300.1, WP_064816095.1,ARV46326.1) or other Bacillus species (e.g., B. inaquosorum(WP_268286106.1 and WP_268353708.1); B. vallismortis (WP_268529903.1 andWP_010328836.1), B. tequilensis (WP_024714271.1 and WP_174227748.1), B.halotolerans (UQZ47588.1), B. atrophaeus (WP_268477846.1 andWP_219946724.1), B. velezensis (WP_182274688.1 and WP_277507204.1), B.amyloliquefaciens (WP_151140090.1) or other suitable organism.

An alsD gene can encode a polypeptide as shown in UniProt Q04777:

SEQ ID NO: 71MKRESNIQVL SRGQKDQPVS QIYQVSTMTS LLDGVYDGDF ELSEIPKYGD FGIGTENKLD GELIGEDGEF YRLRSDGTAT PVQNGDRSPF CSFTFFTPDM THKIDAKMTR EDFEKEINSM LPSRNLFYAI RIDGLFKKVQ TRTVELQEKP YVPMVEAVKT QPIFNEDNVR GTIVGELTPA YANGIAVSGY HLHEIDEGRN SGGHVEDYVL EDCTVTISQK MNMNLRLPNT ADFFNANLDN PDFAKDIETT EGSPE 

In an aspect an alsD polynucleotide can encode a polypeptide having 70,75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQ IDNO:71. In other aspects an alsD gene can encode AIsD polypeptidescomprising 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequenceidentity to AIsD polypeptides from B. subtilis (e.g., GenBank AccessionNumber WP_283933836.1, WP_129134081.1, WP_232920039.1 andWP_136653839.1) or other Bacillus species (e.g., B. spizizenii(MCY7828602.1), B. cabrialesii (WP_129507389.1), B. halotolerans(WP_059353667.1), B. mojavensis (WP_268471631.1), B. tequilensis(WP_167873487.1), B. atrophaeus (WP_061669143), B. nakamurai(WP_061522606.1), B. amyloliquefaciens (WP_071347481.1), B. velezensis(WP_265624765.1)) or other suitable organism).

Malate Dehydrogenase (Mdh3)

One or more malate dehydrogenase (mdh3) polynucleotides can begenetically engineered into a target yeast, e.g., Saccharomyces, so thatthe yeast can express malate dehydrogenase (Mdh3). In an aspect, thegenes encoding the proteins can be obtained from Saccharomyces, such asS. cerevisiae or other suitable organism. A mdh3 gene can encode apolypeptide as shown in UniProt P32419:

SEQ ID NO: 72MVKVAILGAS GGVGQPLSLL LKLSPYVSEL ALYDIRAAEG IGKDLSHINT NSSCVGYDKD SIENTLSNAQ VVLIPAGVPR KPGLTRDDLF KMNAGIVKSL VTAVGKFAPN ARILVISNPV NSLVPIAVET LKKMGKFKPG NVMGVTNLDL VRAETFLVDY LMLKNPKIGQ EQDKTTMHRK VTVIGGHSGE TIIPIITDKS LVFQLDKQYE HFIHRVQFGG DEIVKAKQGA GSATLSMAFA GAKFAEEVLR SFHNEKPETE SLSAFVYLPG LKNGKKAQQL VGDNSIEYFS LPIVLRNGSV VSIDTSVLEK LSPREEQLVN TAVKELRKNI EKGKSFILDS SKL 

In an aspect the last three amino acids are deleted to make tMdh3 (SEQID NO:62). In an aspect an mdh3 or tmdh3 polynucleotide can encode apolypeptide having 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or moresequence identity to SEQ ID NO:72 or SEQ ID NO:62. In other aspects anmdh3 or tmdh3 gene can encode Mdh3 or tMdh polypeptides comprising 70,75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to Mdh3polypeptides from S. cerevisiae (e.g., CA14819955 or EGA62752.1) orother Saccharomyces species (e.g., S. paradoxus (XP_033765191.1), S.mikatae (XP_056080942.1), S. kudriavzevii ((EHN03309.1), S. arboricola(EJS44372.1)) or other suitable organism. The tMdh3 polypeptides willhave the last 3 amino acids deleted before comparison to these or otherMdh3 sequences.

Malic Enzyme

One or more malic enzyme (ME1) polynucleotides can be geneticallyengineered into a target yeast, e.g., Saccharomyces, so that the yeastcan express malic enzyme (Me1). In an aspect, an ME1 polynucleotidecomprises SEQ ID NO:66 or SEQ ID NO:67. In an aspect, an ME1polynucleotide comprises 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or moresequence identity to SEQ ID NO:66 and SEQ ID NO:67. In an aspect, thegenes encoding the protein can be obtained from Rhodosporidium, e.g.,Rhodosporidium toruloides or other suitable organism.

An ME1 gene can encode a polypeptide as shown in UniProt A0A191UMV6

SEQ ID NO: 73MPSTFAPSQP LQGGPSPSQL GPKELLIERA LTRLRSIPSD LEKYTFLAGL RCRNPDVFYG LVGGNMKECC PIIYTPVIGL ACQNWSLIHP PPPESDPTIE ALYLSYSDLP NLPSLIKGLK TRLPHNQMQI SVVTDGSRVL GLGDLGVGGM GISQGKLSLY VAAGGVNPKA TLPIAIDFGT DNEKLLADPL YVGQRMRRLS EEKCLEFMDV FMRCMHETFP NMVIQHEDWQ TPLAFPLLHK NRDLYPCEND DIQGTGAVVL AGAIRAFHLN GVALKDQKIL FFGAGSSGVG VAETICKYFE LQGMSEQEAK SKFWLVDSKG LVAHNRGDTL PSHKKYLARS EPDAPKLRSL KEVVEHVQPT ALLGLSTVGG TFTKEILESM ATYNKRPIVF ALSNPVAQAE CTFEEAIEGT DGRVLYASGS PFDPVEYKEK RYEPGQGNNM YIFPGLGIGA ILARVSKIPE ELVHASAQGL ADSLTPEETA RHLLYPDIER IREVSIKIAV TVIQAAQKLG VDRNEELRGK SSAEIEAYVR KGMYHPLLEA EQQAQ 

In an aspect an ME1 polynucleotide can encode a polypeptide having 70,75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQ IDNO:73. In other aspects an ME1 gene can encode Me1 polypeptidescomprising 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity toMe1 polypeptides from Rhodosporidium toruloides (e.g., GenBank Accessionnumbers KAJ8292376.1, XP_016271116.1, GEM12284.1 or KAJ8292375.1) orother Rhodosporidium species (e.g., R. mucilaginosa (KAG0653902.1) orother suitable organism.

Xylose Reductase

In an aspect, a recombinant yeast can ferment xylose. Where a yeastcannot ferment xylose or it desired that a yeast ferment xylose at abetter rate, then one more recombinant nucleic acid molecules encodingone or more of Xly1, Xly2, and/or Xyl3 can be added to a yeast.

One or more xylose reductase (XYL1) polynucleotides can be geneticallyengineered into a target yeast, e.g., Saccharomyces, so that the yeastcan express xylose reductase (Xyl1). In an aspect, the genes encodingthe protein can be obtained from Scheffersomyces, e.g., Scheffersomycesstipitis or other suitable organism.

An XYL1 gene can encode a polypeptide as shown in UniProt P31867

SEQ ID NO: 74MPSIKLNSGY DMPAVGFGCW KVDVDTCSEQ IYRAIKTGYR LEDGAEDYAN EKLVGAGVKK AIDEGIVKRE DLFLTSKLWN NYHHPDNVEK ALNRTLSDLQ VDYVDLFLIH FPVTFKFVPL EEKYPPGFYC GKGDNEDYED VPILETWKAL EKLVKAGKIR SIGVSNFPGA LLLDLLRGAT IKPSVLQVEH HPYLQQPRLI EFAQSRGIAV TAYSSFGPQS FVELNQGRAL NTSPLFENET IKAIAAKHGK SPAQVLLRWS SQRGIAIIPK SNTVPRLLEN KDVNSFDLDE QDFADIAKLD INLRENDPWD WDKIPIFV 

In an aspect an XYL1 polynucleotide can encode a polypeptide having 65,70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQID NO:74. In other aspects an XYL1 gene can encode Xyl1 polypeptidescomprising 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequenceidentity to Xyl1 polypeptides from Scheffersomyces stipitis (e.g.,GenBank Accession numbers 5Z6T_A or XP_001385181.1) or otherScheffersomyces species (e.g., S. shehatae (Q9P430.1), or other suitableorganism (e.g., Yamadazyma tenuis (074237.1) or Spathaspora roraimensis(ALP00842.1)).

Xylitol Dehydrogenase

One or more xylitol dehydrogenase (XYL2) polynucleotides can begenetically engineered into a target yeast, e.g., Saccharomyces, so thatthe yeast can express xylitol dehydrogenase (Xyl2). In an aspect, thegenes encoding the protein can be obtained from Scheffersomyces, e.g.,Scheffersomyces stipitis or other suitable organism.

An XYL2 gene can encode a polypeptide as shown in UniProt P22144

SEQ ID NO: 75MTANPSLVLN KIDDISFETY DAPEISEPTD VLVQVKKTGI CGSDIHFYAH GRIGNFVLTK PMVLGHESAG TVVQVGKGVT SLKVGDNVAI EPGIPSRESD EYKSGHYNLC PHMAFAATPN SKEGEPNPPG TLCKYFKSPE DFLVKLPDHV SLELGALVEP LSVGVHASKL GSVAFGDYVA VEGAGPVGLL AAAVAKTEGA KGVIVVDIED NKLKMAKDIG AATHTENSKT GGSEELIKAF GGNVPNVVLE CTGAEPCIKL GVDAIAPGGR FVQVGNAAGP VSFPITVEAM KELTLEGSER YGENDYKTAV GIFDTNYQNG RENAPIDFEQ LITHRYKFKD AIEAYDLVRA GKGAVKCLID  GPE 

In an aspect an XYL2 polynucleotide can encode a polypeptide having 65,70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQID NO:75. In other aspects an XYL2 gene can encode Xyl2 polypeptidescomprising 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequenceidentity to Xyl2 polypeptides from Scheffersomyces stipitis (e.g.,GenBank Accession numbers 7Y9P_A or XP_001386982.1) or otherScheffersomyces species (e.g., S. shehatae (AC101079.1), or othersuitable organism (e.g., Spathaspora passalidarum (XP_007373266.1),Spathaspora girioi (ANG59282.1), or Suhomyces tanzawaensis(XP_020064684.1)).

Xylulokinase

One or more xylulokinase (XYL3) polynucleotides can be geneticallyengineered into a target yeast, e.g., Saccharomyces, so that the yeastcan express xylulokinase (Xyl3). In an aspect, the genes encoding theprotein can be obtained from Scheffersomyces, e.g., Scheffersomycesstipitis or other suitable organism.

An XYL3 gene can encode a polypeptide as shown in UniProt Q9P938

SEQ ID NO: 76MTANPSLVLN KIDDISFETY DAPEISEPTD VLVQVKKTGI CGSDIHFYAH GRIGNEVLTK MTTTPEDAPD KLFLGFDLST QQLKIIVTDE NLAALKTYNV EFDSINSSVQ KGVIAINDEI SKGAIISPVY MWLDALDHVF EDMKKDGEPF NKVVGISGSC QQHGSVYWSR TAEKVLSELD AESSLSSQMR SAFTEKHAPN WQDHSTGKEL EEFERVIGAD ALADISGSRA HYRETGLQIR KLSTREKPEK YNRTARISLV SSFVASVLLG RITSIEEADA CGMNLYDIEK REFNEELLAI AAGVHPELDG VEQDGEIYRA GINELKRKLG PVKPITYESE GDIASYFVTR YGENPDCKIY SFTGDNLATI ISLPLAPNDA LISLGTSTTV LIITKNYAPS SQYHLFKHPT MPDHYMGMIC YCNGSLAREK VRDEVNEKEN VEDKKSWDKF NEILDKSTDE NNKLGIYFPL GEIVPNAAAQ IKRSVLNSKN EIVDVELGDK NWQPEDDVSS IVESQTLSCR LRTGPMLSKS GDSSASSSAS PQPEGDGTDL HKVYQDLVKK FGDLFTDGKK QTFESLTARP NRCYYVGGAS NNGSIIXKMG SILAPVNGNY KVDIPNACAL GGAYKASWSY ECEAKKEWIG YDQYINRLFE VSDEMNSFEV KDKWLEYANG VGMLAKMESE LKH

In an aspect an XYL3 polynucleotide can encode a polypeptide having 65,70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQID NO:76. In other aspects an XYL3 gene can encode Xyl3 polypeptidescomprising 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequenceidentity to Xyl3 polypeptides from Scheffersomyces stipitis (e.g.,GenBank Accession numbers AAF72328.2, XP_001387325.2, KAG2731686.1) orother suitable organism (e.g., Spathaspora hagerdaliae (ANG59283.1),Suhomyces tanzawaensis (XP_020063646.1), or Spathaspora gorwiae(ANG59284.1)).

Butanediol Dehydrogenase (BDH1)

One or more butanediol dehydrogenase (BDH1) polynucleotides can begenetically engineered into a target yeast, e.g., Saccharomyces, so thatthe yeast can express butanediol dehydrogenase (Bdh1). In an aspect, thegenes encoding the protein can be obtained from Saccharomyces, e.g.,Saccharomyces cerevisiae or other suitable organism.

A BDH1 gene can encode a polypeptide as shown in UniProt P39714

SEQ ID NO: 77MRALAYFKKG DIHFTNDIPR PEIQTDDEVI IDVSWCGICG SDLHEYLDGP IFMPKDGECH KLSNAALPLA MGHEMSGIVS KVGPKVTKVK VGDHVVVDAA SSCADLHCWP HSKFYNSKPC DACQRGSENL CTHAGFVGLG VISGGFAEQV VVSQHHIIPV PKEIPLDVAA LVEPLSVTWH AVKISGEKKG SSALVLGAGP IGLCTILVLK GMGASKIVVS EIAERRIEMA KKLGVEVENP SKHGHKSIEI LRGLTKSHDG FDYSYDCSGI QVTFETSLKA LTFKGTATNI AVWGPKPVPF QPMDVTLQEK VMTGSIGYVV EDFEEVVRAI HNGDIAMEDC KQLITGKQRI EDGWEKGFQE LMDHKESNVK ILLTPNNHGE MK 

In an aspect a BHD1 polynucleotide can encode a polypeptide having 65,70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to SEQID NO:77. In other aspects a BHD1 gene can encode Bhd1 polypeptidescomprising 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequenceidentity to Bhd1 polypeptides from Saccharomyces cerevisiae (e.g.,GenBank Accession numbers AJ093650.1, GMC37225.1, AJ094666.1,AJ095455.1) or other Saccharomyces species (e.g., S. paradoxus(XP_033764415.1), S. mikatae (XP_056080173.1), S. kudriavzevii(XP_056085817.1), S. uvarum (CA14055538.1, WBF10789.1), S. eubayanus(XP_018224053.1) or other suitable organism.

Deleted or Non-Functional Polynucleotides

In several aspects the yeast described herein have has one or more of adeleted or non-functional GPD1, GPD2, PDC1, ADH1, PHO13, and/or ALD6gene. Deleted, non-functional, or eliminated gene or polynucleotideexpression can be gene or polynucleotide expression that is eliminatedor reduced to an amount that is insignificant or undetectable. Deleted,non-functional, or eliminated gene or polynucleotide expression can alsobe gene or polynucleotide expression that results in an RNA or proteinthat is nonfunctional, for example, deleted gene or polynucleotideexpression can be gene or polynucleotide expression that results in atruncated RNA and/or polypeptide that has substantially no biologicalactivity.

In an embodiment, a genetically engineered or recombinant yeast has noexpression of a polynucleotide encoding one or more of Gpd1, Gpd2, Pdc1,Adh1, Pho13, Ald6 or combinations are present in the yeast. In anaspect, a genetically engineered or recombinant yeast has 50, 60, 70,80, 90, 95, 99% or less expression of a polynucleotide encoding Gpd1,Gpd2, Pdc1, Adh1, Pho13, Ald6, or combinations thereof as compared to awild-type yeast.

The lack of expression can be caused by at least one gene disruption ormutation of a GPD1, GPD2, PDC1, ADH1, PHO13, ALD6 gene or combinationsthereof, which results in no expression of the GPD1, GPD2, PDC1, ADH1,PHO13, ALD6 gene or combinations thereof. For example, the lack ofexpression can be caused by a gene disruption in a GPD1, GPD2, PDC1,ADH1, PHO13, ALD6 gene or combinations thereof which results inattenuated or eliminated expression of the GPD1, GPD2, PDC1, ADH1,PHO13, ALD6 genes or combinations thereof such that the genes can betranscribed but not translated, or the genes can be transcribed andtranslated, but the resulting Gpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6polypeptides or combinations thereof, have substantially no biologicalactivity.

In an embodiment, a recombinant microorganism is mutated or otherwisegenetically altered such that there is substantially no expression ofGpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6 polypeptides in the yeast. In anembodiment, a recombinant yeast is mutated or otherwise geneticallyaltered such that there is substantially no expression of Gpd1, Gpd2,Pdc1, Adh1, Pho13, Ald6 polypeptides thereof in the cell.

In an aspect, a genetically engineered or recombinant yeast has 50, 60,70, 80, 90, 95, 99% or less expression of a Gpd1, Gpd2, Pdc1, Adh1,Pho13, Ald6 polypeptide, or combinations thereof as compared to awild-type microorganism.

The polynucleotides encoding a Gpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6polypeptide or combinations thereof can be deleted or mutated using anysuitable genetic manipulation technique selected from, for example,TALEN, Zinc Finger Nucleases, and CRISPR-Cas9.

One or more regulatory elements controlling expression of thepolynucleotides encoding a Gpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6polypeptide, or combinations thereof can be mutated or replaced toprevent or attenuate expression of a Gpd1, Gpd2, Pdc1, Adh1, Pho13, Ald6polypeptide or combinations thereof, as compared to a control orwild-type yeast. For example, a promoter can be mutated or replaced suchthat the gene expression or polypeptide expression is attenuated or suchthat one or more of GPD1, GPD2, PDC1, ADH1, PHO13, ALD6 genes are nottranscribed. In one embodiment, one or more promoters for a GPD1, GPD2,PDC1, ADH1, PHO13, ALD6 gene or combinations thereof are replaced with apromoter that has weaker activity than the wild-type promoter. Apromoter with weaker activity transcribes the polynucleotide at a rateabout 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% less than the wild-typepromoter for that polynucleotide. In another embodiment, one or morepromoters for GPD1, GPD2, PDC1, ADH1, PHO13, ALD6 genes or combinationsthereof are replaced with an inducible promoter (e.g., TetO promoterssuch as TetO3, TetO7, and CUP1p; P_(GAL1), P_(GAL10), and PGAL7) thatcan be controlled to attenuate expression of the GPD1, GPD2, PDC1, ADH1,PHO13, ALD6 gene or combinations thereof.

The reduced expression, non-expression, or expression of mutated,inactive, or reduced activity polypeptides can be affected by deletionof the polynucleotide or gene encoding Gpd1, Gpd2, Pdc1, Adh1, Pho13,and/or Ald6 polypeptide, replacement of the wild-type polynucleotide orgene with mutated forms, deletion of a portion of a GPD1, GPD2, PDC1,ADH1, PHO13, ALD6 genes or combinations thereof to cause expression ofan inactive form of the polypeptides, or manipulation of the regulatoryelements (e.g. promoter) to prevent or reduce expression of wild-typeGpd1, Gpd2, Pdc1, Adh1, Pho13, and/or Ald6 polypeptides. The promotercould also be replaced with a weaker promoter or an inducible promoterthat leads to reduced expression of the polypeptides. Any method ofgenetic manipulation that leads to a lack of, or reduced expressionand/or activity of Gpd1, Gpd2, Pdc1, Adh1, Pho13, and/or Ald6polypeptides and can be used, including expression of inhibitor RNAs(e.g. shRNA, siRNA, and the like).

Wild-type refers to a yeast that is naturally occurring or which has notbeen recombinantly modified. A control yeast is a yeast that lacksgenetic modifications of a test yeast and that can be used to testaltered biological activity of genetically modified yeast.

A gene disruption is a genetic alteration in a polynucleotide or genethat renders an encoded gene product (e.g., Gpd1, Gpd2, Pdc1, Adh1,Pho13, and/or Ald6 polypeptide) inactive or attenuated (e.g., producedat a lower amount, e.g. about 50, 60, 70, 80, 90, 95, 99% or more loweramount as compared to wild-type or having lower biological activity e.g.about 50, 60, 70, 80, 90, 95, 99% or more lower biological activity ascompared to wild-type). A gene disruption can include a disruption in apolynucleotide or gene that results in no expression of an encoded geneproduct, reduced expression of an encoded gene product, or expression ofa gene product with reduced or attenuated biological activity. Thegenetic alteration can be, for example, deletion of the entire gene orpolynucleotide, deletion of a regulatory element required fortranscription or translation of the polynucleotide or gene, deletion ofa regulatory element required for transcription or translation or thepolynucleotide or gene, addition of a different regulatory elementrequired for transcription or translation or the gene or polynucleotide,deletion of a portion (e.g. 1, 2, 3, 6, 9, 21, 30, 60, 90, 120 or morenucleic acids) of the gene or polynucleotide, which results in aninactive or partially active gene product, replacement of a gene'spromoter with a weaker promoter, replacement or insertion of one or moreamino acids of the encoded protein to reduce its activity, stability, orconcentration, or inactivation of a gene's transactivating factor suchas a regulatory protein. A gene disruption can include a null mutation,which is a mutation within a gene or a region containing a gene thatresults in the gene not being transcribed into RNA and/or translatedinto a functional gene product. An inactive gene product has nobiological activity.

Zinc-finger nucleases (ZFNs), Talens, and CRISPR-Cas9 allow doublestrand DNA cleavage at specific sites in yeast chromosomes such thattargeted gene insertion or deletion can be performed (Shukla et al.,2009, Nature 459:437-441; Townsend et al., 2009, Nature 459:442-445).This approach can be used to modify the promoter of endogenous genes orthe endogenous genes themselves to modify expression of Gpd1, Gpd2,Pdc1, Adh1, Pho13, and/or Ald6 polypeptides, which can be present in theyeast genome. ZFNs, Talens or CRISPR/Cas9 can be used to change thesequences regulating the expression of the polypeptides to increase ordecrease the expression or alter the timing of expression beyond thatfound in a non-engineered or wild-type yeast, or to delete the wild-typepolynucleotide, or replace it with a deleted or mutated form to alterthe expression and/or activity of Gpd1, Gpd2, Pdc1, Adh1, Pho13, and/orAld6 polypeptide.

Yeast Cultures for Production of 2,3-BDO

In an aspect a yeast culture comprising two or more of the recombinantyeast described herein is provided. For example, a yeast culture cancomprise a recombinant yeast, e.g., Saccharomyces such as S. cerevisiae,comprising one or more of a deleted or non-functional GPD1, GPD2, PDC1,ADH1, PHO13, and/or ALD6 gene or polynucleotide. In an aspect, a yeastculture can comprise a recombinant yeast, e.g., Saccharomyces such as S.cerevisiae, comprising one or more of recombinant nucleic acid moleculesencoding alsS, alsD, mdh3, tmdh3, ME1, XYL1, XYL2, XYL3, and/or BDH1. Inan aspect, one or more of PYC1, PYC2, mdh3, tmdh3, and/or BDH1polynucleotides or genes are overexpressed.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalGPD1 polynucleotide or gene such that glyceraldehyde-3-phosphatedehydrogenase (Gpd1) is produced at a lower rate than a wild-type yeasthaving a functional GPD1 gene. In an aspect Gpd1 is produced orexpressed at a rate or amount 20, 30, 40, 50, 60, 70 80, 90, 95, 99%less than a wild-type yeast having a functional GPD1 gene.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalGPD2 polynucleotide or gene such that glyceraldehyde-3-phosphatedehydrogenase (Gpd2) is produced at a lower rate than a wild-type yeasthaving a functional GPD2 gene. In an aspect Gpd2 is produced orexpressed at a rate or amount 20, 30, 40, 50, 60, 70 80, 90, 95, 99%less than a wild-type yeast having a functional GPD2 gene.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalPDC1 polynucleotide or gene such that pyruvate decarboxylase (Pdc1) isproduced at a lower rate than a wild-type yeast having a functional PDC1gene. In an aspect Pdc1 is produced or expressed at a rate or amount 20,30, 40, 50, 60, 70 80, 90, 95, 99% less than a wild-type yeast having afunctional PDC1 gene.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalPDC2 polynucleotide or gene such that pyruvate decarboxylase (Pdc2) isproduced at a lower rate than a wild-type yeast having a functional PDC2gene. In an aspect Pdc2 is produced or expressed at a rate or amount 20,30, 40, 50, 60, 70 80, 90, 95, 99% less than a wild-type yeast having afunctional PDC1 gene.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalADH1 polynucleotide or gene such that alcohol dehydrogenase (Adh1) isproduced at a lower rate than a wild-type yeast having a functional ADH1gene. In an aspect Adh1 is produced or expressed at a rate or amount 20,30, 40, 50, 60, 70 80, 90, 95, 99% less than a wild-type yeast having afunctional ADH1 gene.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalPHO13 polynucleotide or gene such that 4-nitrophenylphosphatase (Pho13)is produced at a lower rate than a wild-type yeast having a functionalPHO13 gene. In an aspect Pho13 is produced or expressed at a rate oramount 20, 30, 40, 50, 60, 70 80, 90, 95, 99% less than a wild-typeyeast having a functional PHO13 gene.

A yeast, e.g., Saccharomyces can comprise a deleted or non-functionalALD6 polynucleotide or gene such that cytosolic aldehyde dehydrogenase(Ald6) is produced at a lower rate than a wild-type yeast having afunctional ALD6 gene. In an aspect Ald6 is produced or expressed at arate or amount 20, 30, 40, 50, 60, 70 80, 90, 95, 99% less than awild-type yeast having a functional ALD6 gene.

A recombinant yeast, e.g., Saccharomyces can comprise additional copies(e.g., 1, 2, 3 or more) of a polynucleotide encoding pyruvatedecarboxylase (Pyc1), pyruvate decarboxylase (Pyc), malate dehydrogenase(Mdh3) (tMdh3), and/or butanediol dehydrogenase (Bdh1), which can be ona plasmid or integrated into the chromosome and overexpressed using astrong promoter, e.g., a strong constitutive promoter. A promoter can beoperably linked to the polynucleotide or gene to be expressed. A strongpromoter can be a P_(TEF1) promoter (Translational elongation factorEF-1 alpha promoter) or a P_(PGK1) promoter (3-phosphoglycerate kinasepromoter). Other strong yeast promoters include, for example, P_(ADH2),P_(TEF2), P_(SSA1), P_(TDH3), P_(PGK1), P_(TPI1), P_(CCW12), andP_(ENO2), P_(GAL1), P_(GAL2), P_(GAL7) and P_(GAL10). Other suitablepromoters are provided in Tang et al., Promoter Architecture andPromoter Engineering in Saccharomyces cerevisiae. Metabolites. 2020 Aug.6; 10(8):320, which is incorporated by reference herein in its entirety.

These polynucleotides can be codon optimized and/or inserted into thechromosome by replacing a gene.

In an aspect, a recombinant yeast, e.g., Saccharomyces can overexpressone or more polypeptides such as pyruvate decarboxylase (Pyc1), pyruvatedecarboxylase (Pyc), malate dehydrogenase (Mdh3) (tMdh3), and/orbutanediol dehydrogenase (Bdh1), using a strong promoter, e.g., a strongconstitutive promoter. In an aspect a mdh3 or tmdh3 gene is operablylinked to a strong P_(TDH3) promoter.

Methods of Production of 2,3-BDO

Methods of producing 2,3-butanediol (2,3-BDO) are provided herein. Anyof the recombinant yeast described herein can be contacted a substrate.The recombinant yeast are allowed to ferment the substrate and 2,3-BDOcollected from the fermentation broth. The yeast cultures and substratescan be fermented using a fed-batch process, a batch process, or anyother suitable process.

A substrate can be lignocellulosic or cellulosic feedstock.Lignocellulosic and cellulosic feedstocks include crop residues likecorn stover, wood residues (e.g., logging residues and forest thinning),dedicated energy crops (e.g., switchgrass, miscanthus, energy cane,sweet sorghum, high biomass sorghum, hybrid poplars, and shrub willows),algae, and industrial and other wastes (e.g., the non-recyclable organicportion of municipal solid waste, biosolids, sludges, waste food,plastics, CO2, industrial waste gases, and manure slurries). Thesefeedstocks are composed of cellulose, hemicellulose, and lignin.

A substrate can comprise glycose, xylose, or a combination of glucoseand xylose.

In an aspect substantially no glycerol and/or substantially no ethanolis accumulated in the fermentation broth. Substantially no glyceroland/or substantially no ethanol is considered to be less than 5 g/L ofglycerol or ethanol accumulated in the final fermentation broth. In anaspect less than 10, 7, 5, 4, 3, 2, 1, 0.5 g/L or less ethanol and/orglycerol is accumulated in the fermentation broth.

In an aspect, a cellulosic or lignocellulosic biomass/substrate can bepre-treated with dilute sulfuric acid to hydrolyze most of the xylan anda small amount of glucan into xylose and glucose, respectively. Thepretreated slurry can be treated by enzymatic hydrolysis (using e.g.,cellobiase and cellulase) where most of the glucan is converted toglucose. The hydrolysate can then be concentrated to increase the sugarconcentrations and to remove fermentation inhibitors including aceticacid, furfural, and hydroxymethyl furfural (HMF). Recovery of 2,3-BDOcan be accomplished via found a hybrid extraction-distillation processor a conventional distillation process. Any suitable solvent (e.g.,oleyl alcohol) can be used for recovering 2,3-BDO. The fermentationeffluent can be first distilled to about 100, 200, 250, 300, 400, 500g/L or more 2,3-BDO for a consistent recovery of 2,3-BDO acrossfermentation titers. The total mass flow rate of solvent sent to themulti-stage mixer-settlers can be about 1.0, 1.2, or 1.5 times the massflow rate of water in the feed. The extract is distilled at a pressureof about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or 0.09 atm to operate thereboiler at a temperature (e.g., about 400, 500, 524.77, 550, 600 K) atwhich heating agents are available and to lower the heating requirement.The water, 2,3-BDO, and acetoin are then distilled by sequentialconventional distillation at about 0.1, 0.2, or 0.3 atm. The recoveredacetoin can be sent back to fermentation to increase the overallconversion to 2,3-BDO.

In an aspect, methods described herein can produce 2,3-BDO at more than0.1, 0.5, 1.0, 1.25, 1.5, 1.75 g/L/h. In an aspect, methods describedherein can produce 2,3,-BDO at a yield of 50, 70, 80, 90, 100, 110, 120,130, 140, 150 g/L or more.

Methods of Producing Methyl Ethyl Ketone (MEK)

In an aspect, methods of producing methyl ethyl ketone are provided. Asdiscussed above, a substrate can be contacted a recombinant yeastculture as described herein under fermentation conditions. 2,3-BDO canbe collected from the fermentation broth and purified from to formpurified 2,3-BDO. The purified 2,3-BDO can be subjected to catalyticdehydration such that MEK is produced. A catalyst for the catalyticdehydration can be, for example, tricalcium phosphate or any othersuitable catalyst. The purified 2,3-BDO can be greater than 70, 80, 90,95, 97, 98, 99, 99.5 wt % pure or more.

In an aspect, concentrated hydrolysate can be used as a substrate forcontinuous fermentation to produce crude 2,3-BDO (the fermentationbroth), following a series of solvent extraction and vacuum distillationunit operations which can be used to separate and purify 2,3-BDO toapproximately 100 wt %. The purified 2,3-BDO can subsequently beconverted to MEK (major) and isobutyraldehyde (IBA, minor) as productsvia catalytic dehydration (see, e.g., [6], [11], [40]). Finally, MEK canpurified through vacuum distillation to >99.5 wt % and sold as the mainproduct of the biorefinery, and IBA can be hydrogenated into isobutylalcohol (IBO), which is purified to >99.5 wt % through distillation andsold as a co-product.

In some aspects MEK can be produced by pre-treating cellulosic orlignocellulosic biomass/substrate with dilute sulfuric acid to hydrolyzemost of the xylan and a small amount of glucan into xylose and glucose,respectively. The pretreated slurry can be treated by enzymatichydrolysis (using e.g., cellobiase and cellulase) where most of theglucan is converted to glucose. The hydrolysate can then be concentratedto increase the sugar concentrations and to remove fermentationinhibitors including acetic acid, furfural, and hydroxymethyl furfural(HMF). Hydrolysate concentration can be realized through a multi-effectevaporator (see FIG. 13 ; [6]), which can be included to reduce themaximum concentration of fermentation inhibitors (acetic acid, furfural,and hydroxymethyl furfural, all of which are volatile) to below 1 g/L[5]. The hydrolysate can be concentrated so that at the target titer,the steady state concentration of 2,3-BDO in the main fermenter equalsthe target titer. In the case that not enough inhibitors have beenremoved, the hydrolysate can be concentrated to a greater level thanneeded for the target titer (so that more inhibitors can be removed),and dilution water can be added in the fermenter to maintain the targettiter. Further, a maximum sugar (glucose, xylose, mannose, arabinose,and galactose) concentration of can be about 200, 300, 400, 500, 600,700, 800, or 900 g/L so that the viscosity of the concentratedhydrolysate will not exceed the capacity of the centrifuge pump [5].

For the fermentation units (a main fermenter and a seed train), thedesign algorithms as in Bhagwat et al. [5] can be used to size thereactors (based on the retention time calculated through target titerand productivity).

Recovery of 2,3-BDO can be accomplished via found a hybridextraction-distillation process (Harvianto et al. [9]) or a conventionaldistillation process. Any suitable solvent (e.g., oleyl alcohol) can beused for recovering 2,3-BDO. The fermentation effluent can be firstdistilled to about 100, 200, 250, 300, 400, 500 g/L or more 2,3-BDO fora consistent recovery of 2,3-BDO across fermentation titers. The totalmass flow rate of solvent sent to the multi-stage mixer-settlers can beabout 1.0, 1.2, or 1.5 times the mass flow rate of water in the feed.The extract is distilled at a pressure of about 0.03, 0.04, 0.05, 0.06,0.07, 0.08, or 0.09 atm to operate the reboiler at a temperature (e.g.,about 400, 500, 524.77, 550, 600 K) at which heating agents areavailable and to lower the heating requirement. The water, 2,3-BDO, andacetoin are then distilled by sequential conventional distillation atabout 0.1, 0.2, or 0.3 atm. The recovered acetoin can be sent back tofermentation to increase the overall conversion to 2,3-BDO.

The purified (around 99.7 wt %) 2,3-BDO stream can then sent tocatalytic dehydration at about 200, 250, 300, 325, or 350° C. with acatalyst such as tricalcium phosphate. During the reaction, 2,3-BDO isconverted to MEK and to isobutyraldehyde (IBA) [10]. After the reaction,the reactant stream is passed through a distillation column with IBAseparated as the top product and MEK and 2,3-BDO in the bottom product.IBA can then be hydrogenated to isobutyl alcohol (IBO) using, e.g., akieselguhr-supported carboxymethylcellulose-nickel catalyst or othersuitable catalyst such that a >99.5 wt % pure IBO product can beobtained by removing the unreacted IBA using another distillationcolumn, and the removed IBA can be recycled back to the hydrogenationreactor. Finally, the bottom product containing MEK and unreacted2,3-BDO can be sent to another distillation column, where a MEK productof >99.9 wt % purity can be obtained through vacuum distillation, andthe unreacted 2,3-BDO can be recycled back to the dehydration reactor.

Methods of Inducing Drought Tolerance and Improving Plant Health

In an aspect, drought tolerance can be induced in plants by contactingroots of the plants (e.g., monocotyledons or dicotyledons) with a spentfermentation broth. A fermentation broth can be produced by contactingany yeast culture described herein with a fermentation medium orsubstrate. The fermentation broth is the fluid remaining after afermentation reaction.

In an aspect, plant health can be improved by contacting roots of theplants (e.g., monocotyledons or dicotyledons) with a spent fermentationbroth. A spent fermentation broth can be the result of any medium thathas been fermented by any yeast culture described herein. A fermentationbroth can be used without any additional purification steps. In anaspect, a fermentation broth can be partially purified to remove yeastcells and fermentation debris. In an aspect, a fermentation broth can bepartially purified to increase the purity of the 2,3-BDO. In an aspect,a fermentation broth or medium has substantially no glycerol and/orsubstantially no ethanol. Substantially no glycerol and/or substantiallyno ethanol is considered to be less than 5 g/L of glycerol or ethanolaccumulated in the final fermentation broth or medium. In an aspect lessthan 10, 7, 5, 4, 3, 2, 1, 0.5 g/L or less ethanol and/or glycerol ispresent in the final fermentation broth or medium.

In an aspect, a fermentation broth comprises about 100, 200, 250, 300,400, 500, 600, 700 or more μM or 2,3-BDO.

In an aspect, plants subjected to drought conditions and treated with afermentation medium as described herein have a survival rate at least1.1., 1.5, 1.75, 2.0, 2.5, 3.0, 4.0, 5.0-fold or more higher than plantsnot contacted with the fermentation medium.

The compositions and methods are more particularly described below andthe Examples set forth herein are intended as illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art. The terms used in the specification generally havetheir ordinary meanings in the art, within the context of thecompositions and methods described herein, and in the specific contextwhere each term is used. Some terms have been more specifically definedherein to provide additional guidance to the practitioner regarding thedescription of the compositions and methods.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. As used in the descriptionherein and throughout the claims that follow, the meaning of “a”, “an”,and “the” includes plural reference as well as the singular referenceunless the context clearly dictates otherwise. The term “about” inassociation with a numerical value means that the value varies up ordown by 5%. For example, for a value of about 100, means 95 to 105 (orany value between 95 and 105).

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by referenceherein in their entirety. The embodiments illustratively describedherein suitably can be practiced in the absence of any element orelements, limitation or limitations that are specifically or notspecifically disclosed herein. Thus, for example, in each instanceherein any of the terms “comprising,” “consisting essentially of,” and“consisting of” can be replaced with either of the other two terms,while retaining their ordinary meanings. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claims. Thus, itshould be understood that although the present methods and compositionshave been specifically disclosed by embodiments and optional features,modifications and variations of the concepts herein disclosed can beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of the compositions andmethods as defined by the description and the appended claims.

Any single term, single element, single phrase, group of terms, group ofphrases, or group of elements described herein can each be specificallyexcluded from the claims.

Whenever a range is given in the specification, for example, atemperature range, a time range, a composition, or concentration range,all intermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the aspects herein. It will be understoodthat any elements or steps that are included in the description hereincan be excluded from the claimed compositions or methods

In addition, where features or aspects of the compositions and methodsare described in terms of Markush groups or other grouping ofalternatives, those skilled in the art will recognize that thecompositions and methods are also thereby described in terms of anyindividual member or subgroup of members of the Markush group or othergroup.

The following are provided for exemplification purposes only and are notintended to limit the scope of the embodiments described in broad termsabove.

EXAMPLES Example 1 Materials and Methods

Strains and Media

E. coli Top10 [F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80/acZΔM15 ΔlacX74 recA1araD139 Δ(ara-leu) 7697 ga/U ga/K rpsL (Str^(R)) endA1 nupG] was usedfor manipulation of plasmids. The E. coli strains were grown in LuriaBertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl) at 37° C.with ampicillin (100 μg/mL) if necessary. A xylose-fermenting S.cerevisiae CT2 strain-a D452-2 derived strain with integration of twocopies of expression cassettes containing XYL1, XYL2, and XYL3 in thebackground of PHO13 and ALD6 deletion [35]—was used as a host strain forintroducing genetic modifications to produce 2,3-BDO. The CT2 strain,and its derived yeast strains were cultivated at 30° C. in YP medium (10g/L yeast extract, 20 g/L peptone) with 20 g/L of glucose. ForCRISPR-Cas9 based genome editing experiments, 120 μg/mL ofnourseothricin, 300 μg/mL of geneticin, and 300 μg/mL of hygromycin Bwere added as necessary for selecting transformants.

Plasmid and Strain Construction

The strains used in this study are listed in Table 1. The plasmids,primers, guide RNA (gRNA) target sequences, and synthetic DNA sequencesused in this study are listed in Table 2, 3, 4, and 5, respectively.

TABLE 1 Strains used or constructed in this study Strain DescriptionSource CT2 D452-2 pho13Δ::XYL123 ald6Δ::XYL123 his3, Tsai et al. [35]trp1, leu2, and ura3 CTL CT2 pdc1Δ::LDH from Rhizopus oryzae This studyCTLA CT2 pdc1Δ::LDH from Rhizopus oryzae, adh1Δ This study CTLAP CTLA inwhich the endogenous promoter of This study PDC5 gene was substitutedwith TEX1 promoter CTLAB CTLA in which the P_(TDH3)-alsS-T_(CYC1)cassette has This study been integrated on chr XVI and theP_(TDH3)-alsD- T_(CYC1) cassette has been integrated on chr VII CTLABMCTLAB has a mutation on ldhA gene (117^(th) This study amino acid wasmissed) CTLABG1 CTLABM, gpd1Δ This study CTLABG2 CTLABM, gpd2Δ Thisstudy CTLABG1G2 CTLABM, gpd1Δgpd2Δ This study CBMM CTLABG1G2 employing aPyruvate-Malate cycle This study CBMMP1 CBMM in which the endogenouspromoter of This study PYC1 gene was substituted with the TEF1 promoterCBMMP2 CBMM in which the endogenous promoter of This study PYC2 gene wassubstituted with the PGK1 promoter CBMMP1P2 CBMM in which the endogenouspromoter of This study PYC1 gene was substituted with the TEF1 promoterand the endogenous promoter of PYC2 gene was substituted with the PGK1promoter

TABLE 2 Plasmids used in this study Name Description of plasmidsReference pRS423GPD HIS3, TDH3 promoter, CYC1 terminator, 2μ origin,Mumberg et al. [73] and Amp^(R) pRS426GPD URA3, TDH3 promoter, CYC1terminator, 2μ origin, Mumberg et al. [73] and Amp^(R) pRS426TEF URA3,TEF1 promoter, CYC1 terminator, 2μ origin, This study and Amp^(R)pRS423_alsS pRS423GPD harboring alsS gene from B. subtilis Kim et al.[74] pRS426_alsD pRS426GPD harboring alsD gene from B. subtilis Kim etal. [74] pRS405_LDH pRS405PGK harboring ldhA gene from R. oryzae Turneret al. [75] pRS423_MDH3 pRS423GPD harboring MDH3 gene from This study S.cerevisiae pCas9-NAT Cas9 expression plasmid, NAT1 marker Zhang et al.[57] pRS42K 2μ origin, KanMX EUROSCARF pRS42H 2μ origin, hph EUROSCARFpRS42H-CS6 pRS42H, gRNA cassette targeting the intergenic site Kwak etal. [59] on Chr VII pRS42K-CS8 pRS42K, gRNA cassette targeting theintergenic site Kwak et al. [59] on Chr XVI pRS42K-CS9 pRS42K, gRNAcassette targeting the intergenic site Lee et al. [58] on Chr VIIIpRS42K-PDC1 pRS42K, gRNA cassette targeting the PDC1 gene This studypRS42H-ADH1 pRS42H, gRNA cassette targeting the ADH1 gene This studypRS42K-PDC5 pRS42K, gRNA cassette targeting the promoter of This studythe PDC5 gene pRS42K-GPD1 pRS42K, gRNA cassette targeting the GPD1 geneThis study pRS42H-GPD2 pRS42H, gRNA cassette targeting the GPD2 geneThis study pRS42H-LDH pRS42H, gRNA cassette targeting the LDH gene inThis study the PDC1 locus pRS42K-PYC1 pRS42H, gRNA cassette targetingthe promoter of This study the PYC1 gene pRS42H-PYC2 pRS42H, gRNAcassette targeting the promoter of This study the PYC2 gene

TABLE 3 Primers used in this study Name Direction Sequence GPD-F Sense5′-GCTGCAGGAATTCGATATCAAGCT-3′ (SEQ ID NO: 1) GPD-R Antisense5′-CCGGGGGATCCACTAGTTCTAGAA-3′ (SEQ ID NO: 2) tMDH3-F Sense5′-AAACACCAGAACTTAGTTTCGACGGATTCTAGAACTAGTGGATCCCCCGGATGGTCAAAGTCGCAATTCTTGGC-3′(SEQ ID NO: 3) tMDH3-R Antisense5′-ATGACTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCTTAAGAGTCTAGGATGAAACTCTTGCC-3′ (SEQ ID  NO: 4) gRNA-U Sense5′-CCCGAGCTCTCTTTGAAAAGATAATGTATGATTATG-3′ (SEQ ID NO: 5) gRNA-DAntisense 5′-AACTGCAGGGATCCAGACATAAAAAACAAAAAAAGCAC-3′ (SEQ ID NO: 6)dDNA-LDH-F Sense 5′tcataacctcacgcaaaataacacagtcaaatcaatcaaaATGGTATTACACTCAAAGGT-3′ (SEQ ID NO: 7) dDNA-LDH-R Antisense5′-tgcttataaaactttaactaataattagagattaaatcgcTCCTCAACAGCTACTTTTAG-3′ (SEQ ID NO: 8) dDNA-ADH1-F Sense5′-CTGCACAATATTTCAAGCTATACCA-3′ (SEQ ID NO: 9) dDNA-ADH1-R Antisense5′-CATAAGAAATTCGCTTATTTAGAAGTGT-3′ (SEQ ID NO: 10) dDNA-TEX1-F Sense5′-gccaaggaaataaagcaaataacaataacaccattattttGCATAACCTTGAAGGTTAAC-3′ (SEQ ID NO: 11) dDNA-TEX1-R Antisense5′-tcaatctttcaaataaatatttacctaaggttatttcaGACATGCCGAATAGTTCACTTG-3′ (SEQ ID NO: 12) dDNA-CS6-F Sense5′-aacctcgaggagaagtttttttacccctctccacagatcCAGGAAACAGCTATGACCATG-3′ (SEQ ID NO: 13) dDNA-CS6-R Antisense5′-taattaggtagaccgggtagatttttccgtaaccttggtgtcTGTAAAACGACGGCCAGT-3′ (SEQ ID NO: 14) dDNA-CS8-F Sense5′-caaaattacctacggtaattagtgaaaggccaaaatctaatgttacaataAATTAACCCTCACTAAAGGGA-3′ (SEQ ID NO: 15) dDNA-CS8-R Antisense5′-gaccgttcccttgtgttgtaccagtggtagggttcttctcggtagcttctGTAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 16) dDNA-GPD1-F Sense5′-TTAATTTTCTTTTATCTTACTCTCC-3′ (SEQ ID NO: 17) dDNA-GPD1-R Antisense5′-TAGTTATGAGAAATGACATAATGC-3′ (SEQ ID NO: 18) dDNA-GPD2-F Sense5′-CGCTCCCCTTCCTTATCAATGC-3′ (SEQ ID NO: 19) dDNA-GPD2-R Antisense5′-GGAGAGTGTCTATTCGTCATCG-3′ (SEQ ID NO: 20) dDNA-CS9-F Sense5′-aggattcattagtggaaaagttcagtgacaaaatctagaaaataatatgaAATTAACCCTCACTAAAGGGA-3′ (SEQ ID NO: 21) dDNA-CS9-R Antisense5′-gaatatagcgtatttttatttaatcacggtacaatggagatatttgcatgGTAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 22) dDNA-ME-F Sense5′-tctcaattattattttctactcataacctcacgcaaaataacacagtcaaatcaatcaaaATGCCAGCACATTTTGCCCC (SEQ ID NO: 23) dDNA-ME-R Antisense5′-tatttttcgttacataaaaatgcttataaaactttaactaataattagagattaaatcgcCTACTGTGCCTGCTGTTCCG (SEQ ID NO: 24) dDNA-PYC1-F Sense5′-ccgtacttgcagcccgttgccaattgccgcctaatattgtCATAGCTTCAAAATGTTTCT (SEQ ID NO: 25) dDNA-PYC1-R Antisense5′-tgaagttatctctcaagccggcgaattttctttgcgacatCTTAGATTAGATTGCTATGC (SEQ ID NO: 26) dDNA-PYC2-F Sense5′-cctcaaacaagaattgtacgacattacgttcaagaaaattAAGAAATTACCGTCGCTCGT (SEQ ID NO: 27) dDNA-PYC2-R Antisense5′-aattgtccctaagaccggccaatttcttgctactgctcatAGACATTGTTTTATATTTGT (SEQ ID NO: 28) Conf-LDH-F Sense5′-TTCATAATTGCATAATATTGTCCGC-3′ (SEQ ID NO: 29) Conf-LDH-R Antisense5′-TTGCAATGTGTGTCAAGATATCG-3′ (SEQ ID NO: 30) Conf-ADH1-F Sense5′-TTACACTGCCTCATTGATGGTGG-3′ (SEQ ID NO: 31) Conf-ADH1-R Antisense5′-TACAATTGGGTGAAATGGGGAGCG-3′ (SEQ ID NO: 32) Conf-PDC5-F Sense5′-GGTGAGAATCCTTCTGATGCATACT-3′ (SEQ ID NO: 33) Conf-PDC5-R Antisense5′-GTGCTCTACTGGTGATTTTTCATCG-3′ (SEQ ID NO: 34) Conf-CS6-F Sense5′-GTCTGCCGAAATTCTGTG-3′ (SEQ ID NO: 35) Conf-CS6-R Antisense5′-CGGTCAGAAAGGGAAATG-3′ (SEQ ID NO: 36) Conf-CS8-F Sense5′-AGTGGAACATAGAAGGGG-3′ (SEQ ID NO: 37) Conf-CS8-R Antisense5′-TAAGCAGCCCAGTGAAC-3′ (SEQ ID NO: 38) Conf-GPD1-F Sense5′-CCTACTGTCCCTATGTCTCTGG-3′ (SEQ ID NO: 39) Conf-GPD1-R Antisense5′-CCAAAGTACATCCTTGTCGAGC-3′ (SEQ ID NO: 40) Conf-GPD2-F Sense5′-AAGAGTGTTTAGCTTACGGACCTATTGCCA-3′ (SEQ ID NO: 41) Conf-GPD2-RAntisense 5′-CAGTAGTGACTAACATAGCGCTCTTATCTC-3′ (SEQ ID NO: 42)Conf-CS9-F Sense 5′-TGGTAATGAGGAATGCGT-3′ (SEQ ID NO: 43) Conf-CS9-RAntisense 5′-CGGGCATTATGCGTAGAT-3′ (SEQ ID NO: 44) Conf-ME-F Sense5′-TTCATAATTGCATAATATTGTCCGC-3′ (SEQ ID NO: 45) Conf-ME-R Antisense5′-GACAGTGCAGTAATAATATGAACC-3′ (SEQ ID NO: 46) Conf-PYC1-F Sense5′-TCGACGAATGGTAGCGCTTG-3′ (SEQ ID NO: 47) Conf-PYC1-R Antisense5′-CTTAGATTAGATTGCTATGC-3′ (SEQ ID NO: 48) Conf-PYC2-F Sense5′-CCTCAAACAAGAATTGTACG-3′ (SEQ ID NO: 49) Conf-PYC2-R Antisense5′-AATTGTCCCTAAGACCGGCC-3′ (SEQ ID NO: 50) Restriction sites areunderlined, and homologous regions for Donor DNA integration arelowercased.

TABLE 4 Target sequences of gRNA used for CRISPR-Cas9based genome editing in this study Name Sequence CS65′-GATACTTATCATTAAGAAAA-3′ (SEQ ID NO: 51) CS85′-TGATTCAATCATTCTTATTG-3′ (SEQ ID NO: 52) CS95′-TAACTATTACTTGTTTCTAT-3′ (SEQ ID NO: 53) PDC15′-TCTGTCAATTTCAGCTGGGG-3′ (SEQ ID NO: 54) ADH15′-CCATCTTGTGTGCTGGTATC-3′ (SEQ ID NO: 55) PDC55′-GGAAAAGCCTCCATATCCAA-3′ (SEQ ID NO: 56) GPD15′-GGCTGCCGAAAAGCCTTTCA-3′ (SEQ ID NO: 57) GPD25′-TGCAAACTTGGCACCGGAAG-3′ (SEQ ID NO: 58) LDH5′-AGTCACACGCCCATCCGAGC-3′ (SEQ ID NO: 59) PYC15′-TAGAGGGACCTGTGTTTGAC-3′ (SEQ ID NO: 60) PYC25′-ATTACTATATTGCAAAATAA-3′ (SEQ ID NO: 61)

TABLE 5 Synthetic DNA sequence used in this study Name Sequence (5′→3′)Source tMDH3 ATGGTCAAAGTCGCAATTCTTGGCGCTTCTGGTGGCGTGGG MDH3 geneACAACCGCTATCATTACTGCTAAAATTAAGCCCTTACGTTTCC fromGAGCTGGCGTTGTACGATATCCGAGCTGCGGAAGGCATTGG S. cerevisiaeTAAGGATTTATCTCACATCAACACCAACTCAAGTTGTGTCGG (SEQ IDTTATGATAAGGATAGTATTGAGAACACCTTGTCAAATGCTCA NO: 62)GGTGGTGCTAATACCGGCTGGTGTTCCCAGAAAGCCCGGTTTAACTAGAGATGATTTGTTCAAGATGAACGCCGGTATTGTCAAAAGCCTGGTAACCGCTGTTGGAAAGTTCGCACCAAATGCGAGGATTTTAGTCATTTCAAACCCTGTAAACAGTTTGGTCCCTATTGCTGTGGAAACTTTGAAGAAAATGGGTAAGTTCAAACCTGGAAACGTTATGGGTGTGACGAACCTTGACCTGGTACGTGCA GAAACCTTTTTGGTAGATTATTTGATGCTAAAAAACCCCAAAATTGGACAAGAACAAGACAAAACTACAATGCACAGAAAGGTCA CTGTTATTGGGGGTCATTCAGGGGAAACCATTATCCCAATAATCACCGACAAATCGCTGGTATTTCAACTTGATAAGCAGTACGAGCACTTCATTCATAGGGTCCAGTTCGGAGGTGATGAAATTGTCAAAGCTAAACAGGGCGCCGGTTCCGCCACGTTGTCCATGGCGTTCGCGGGGGCCAAGTTTGCTGAAGAAGTTTTGAGGAGCTTCCATAATGAGAAACCAGAAACGGAGTCACTTTCCGCATTCGTTTATTTACCAGGCTTAAAAAACGGTAAGAAAGCGCAGCAATTAGTTGGCGACAACTCTATTGAGTATTTTTCCTTGCCAATTGTTTTGAGAAATGGTAGCGTAGTATCCATCGATACCAGTGTTCTGGAAAAACTGTCTCCGAGAGAGGAACAACTCGTTAATACTGCGGTCAAAGAGCTACGCAAGAATATTGAAAAAGGCAAGAG TTTCATCCTAGACTCTTCCAAGCTATGADonor CTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTATC ADH1TCATATACAATGTCTATCCCAGAAACTCAAAAAGGTGTTATCT (SEQ IDTCTACGAATCCCACGTCGGCTTGTCTACCTTGCCAGAAATT NO: 63)TACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATG DonorTTAATTTTCTTTTATCTTACTCTCCTACATAAGACATCAAGAAA GPD1CAATTGTATATTGTACACCCCCCCCCTCCACAAACACAAATA (SEQ IDTTGATAATATAAAGATTTATTGGAGAAAGATAACATATCATAC NO: 64)TTTCCCCCACTTTTTTCGAGGCTCTTCTATATCATATTCATAA ATTAGCATTATGTCATTTCTCATAACTADonor CGCTCCCCTTCCTTATCAATGCTTGCTGTCAGAAGATTAACA GPD2AGATACACATTCCTTAAGCGAACGCATCCGGTGTTATATACT (SEQ IDCGTCGTGCATATAAAAATTCGAGGCAGTCTACCAGATAGTCT NO: 65)ACAACAACGTCCGCATGGAAGACCTACCGGAGATGATTGAAGAGCTAGACATCGATGACGAATAGACACTCTCC ME1ATGCCAGCACATTTTGCCCCATCCCAACCATTACAAGGTGG (SEQ IDGCCAAGCCCCAGCCAACTGGGACCAAAAGAACTGCTGATTG NO: 66)AGAGGGCTCTTACCAGACTACGTTCCATCCCCAACGACTTAGAAAAATACACATTTTTAGCGGGGCTAAGAGGAAGAAATCCAGACGTTTTTTATGGATTAGTCGGGGGAAATATGAAAGAGTGTTGCCCGATTATATATACCCCAGTGATAGGGCTTGCCTGTCAAAATTGGTCCTTAATCCATCCGCCTCCCCCTGAATCCGACCCAACGATTGACGCTCTTTACTTGAGTTATAGCGATCTTCCAAACCTACCCCAGCTTATCGGTGGGTTAAAGACAAGGTTACCTCATGATCAGATGCAGATCAGCGTTGTCACTGACGGTAGCCGTGTCCTTGGCTTAGGTGATCTGGGGGTTGGCGGAATGGGGATATCCCAAGGAAAGCTATCACTGTATGTCGCCGCAGGGGGTGTGAATCCTAAAGCCACTTTGCCTATAGCAATTGATTTCGGCACAGATAATGAGACTCTGCTAGCCGATCCGTTGTACGTAGGTCAAAGGATTCGTAGATTGAGCCAGGAGAAGTGCTTAGAGTTCATGGAGGTCTTTATGCGTTGCATGCATGAGACTTTTCCCAATATGGTAATCCAACACGAAGATTGGCAAACTCCGCTGGCCTTCCCTCTATTACACAAAAACCGTGATCTATACCCATGCTTCAACGATGATATCCAAGGAACCGGCGCTGTAGTACTGGCCGGCGCCATAAGAGCATTCCATCTGAACGGCGTCGCACTGAAAGACCAGAAAATTTTGTTCTTTGGCGCGGGGTCAAGTGGCGTGGGTG TCGCGGAGACGATC ME1GGCGCGGGGTCAAGTGGCGTGGGTGTCGCGGAGACGATC ME1 from (SEQ IDTGCAAATACTTTGAGCTACAGGGGATGAGTGAAGATGAAGC R. toruloides NO: 67)TAAAAGCAAATTCTGGTTGGTTGATAGTAAGGGGCTAGTTGC was codonTCACAATCGTGGCGATACTTTACCTAGCCACAAAAAGTACCT optimized forTGCAAGAAGTGAGCCTGATGCGCCTAAATTGAGGACGCTAA S. cerevisiaeAGGAAGTCGTAGAACATGTACAACCCACGGCCTTGTTAGGGCTTAGCACAGTTGGGGGTACATTCACAAAGGAAATTCTTGAAGCAATGGCCACTTATAACAAACGTCCTATTGTCTTTGCACTTAGCAATCCAGTAGCGCAAGCTGAATGCACGTTCGAGGAAGCTGTGGAGGGAACCGACGGAAGGGTCTTATATGCCAGTGGAAGTCCCTTTGACCCCGTGGAGTACAAAGGCAAAAGGTATGAACCAGGACAGGGCAACAATATGTACATCTTCCCCGGCCTGGGTATAGGTGCCATTTTGGCGAGGGTGAGTAAAATCCCGGAAGAGCTGGTTCATGCATCAGCACAAGGGCTTGCGGACTCATTAACGCCGGAGGAAACGGCGCGTCATTTGCTGTATCCAGATATAGAACGTATAAGGGAAGTTAGTATTAAGATAGCAGTTACCGTAATCCAAGCGGCACAGAAGCTTGGTGTTGATAGAAACGAGGAGCTTAGAGGGAAAAGCAGTGCTGAAATTGAAGCCTATGTACGTAAAGGGATGTACCATCCATTACTTGAGGCGGAACAGCA GGCACAGTAG tMDH3 gene from S.cerevisiae was synthesized except for the last 9 base pairs that encodethe peroxisomal targeting sequence (tripeptide SKL)

Recombinant DNA techniques were performed according to standardprocedures. A lithium acetate transformation method with single strandcarrier DNA and polyethylene glycerol [36] was used to introduceCas9-NAT, gRNA expression vectors, and donor DNA fragment into yeaststrains. Putative transformants on selection plates were confirmed bycolony PCR.

Plasmid Construction

Construction of pRS423_MDH3: To express malate dehydrogenase (MDH) inthe cytosol of S. cerevisiae, a truncated MDH3 (tMDH3) gene from S.cerevisiae was synthesized except for the last 9 base pairs (tripeptideSKL) encoding for peroxisomal targeting sequence using the gBlocksservice from Integrated DNA Technologies (IDT, IA, USA). To construct atruncated MDH expression plasmid (pRS423_tMDH3), a DNA fragment (vectorfraction) was amplified from pRS423GPD plasmid using GPD-F and GPD-Rprimers and another DNA fragment (insert fraction) was amplified fromthe synthetic oligomer using tMDH3-F and tMDH3-R primers. The two PCRproducts were ligated together by in vitro homologous recombinationusing a Gibson assembly cloning kit (NEB, MA, USA).

Construction of Cas9-NAT and gRNA plasmids: Cas9-NAT plasmid (Addgeneplasmid #64329) [57] was adopted for the expression of Cas9 nuclease inyeast. gRNA expression cassettes targeting intergenic site on chromosomeVII (CS6), XVI (CS8), and VIII (CS9) were designed by replacing thetarget sequence of previous gRNA cassettes [58, 59]. The gRNA cassetteswere PCR amplified using gRNA-U and gRNA-D primers and inserted into 2-μplasmids pRS42K and pRS42H (EUROSCARF).

Strain Construction

The CTL strain was constructed via deletion of major isozyme of pyruvatedecarboxylase (pdc1Δ) and chromosomal integration of lactatedehydrogenase (IdhA) expression cassette (P_(PGK1)-IdhA-T_(CYC1)) intothe CT2 strain. The IdhA expression cassette was amplified frompRS405_LDH plasmid using primer sets of dDNA-LDH-F and dDNA-LDH-R andintegrated into the PDC1 locus of CT2 strain using CRISPR-Cas9 basedgenetic modification as previously reported [57, 59], followed byselection on YPDNK plate. Colonies were randomly picked from the plateand verified by PCR amplification using primers (Conf-LDH-F andConf-LDH-R).

The CTLA strain was constructed via deletion of one major isozyme ofalcohol dehydrogenase (adh1A) in the CTL strain. Donor DNA containingthe homologous regions of the upstream and downstream of the ADH1 genewas synthesized using the gBlocks service from Integrated DNATechnologies (IDT, IA, USA). The donor DNA was amplified from thesynthesized DNA oligomer using primer sets of dDNA-ADH1-F anddDNA-ADH1-R and integrated into the ADH1 locus of the CTL strain usingCRISPR-Cas9 based genetic modification as previously reported [57, 59],followed by selection on YPDNH plate. Colonies were randomly picked fromthe plate and verified by PCR amplification using primers (Conf-ADH1-Fand Conf-ADH1-R).

The CTLAB strain was constructed via chromosomal integration of one copyof cassette expressing alsS gene and one copy of cassette expressingalsD gene. The alsS expression cassette (P_(TDH3)-alsS-T_(CYC1)) wasamplified from pRS423_alsS plasmid using primer sets of dDNA-CS6-F anddDNA-CS6-R, and the alsD expression cassette (PTDH3-alsD-TcYc1) wasamplified from pRS426_alsD plasmid using primer sets of dDNA-CS8-F anddDNA-CS8-R, respectively. The expression cassettes were integrated intothe intergenic site on chromosome VII (CS6) and XVI (CS8) in the CTLAstrain, respectively, followed by selection on YPDNHK plate. Colonieswere randomly picked from the plate and verified by PCR amplificationusing primers (Conf-CS6-F and Conf-CS6-R, and Conf-CS8-F andConf-CS8-R), respectively.

Strain CTLABM was constructed via spontaneous mutation during the curingof Cas9-NAT and guide RNA plasmids, after the CTLAB strain construction.To cure the Cas9-NAT and guide RNA plasmids, the CTLAB strain wascultured and transferred in YPD20 (YP medium with 20 g/L glucose)several times. After curing the two plasmids, the CTLAB strain exhibitedsignificant phenotypic changes that glycerol was produced instead oflactate with 2,3-BDO as major products. To elucidate the phenotypicchanges, we sequenced the/dhA gene of the CTLAB strain using primer setsof Conf-LDH-F and dDNA-LDH-R. As a result, the 117^(th) (valine) aminoacid, a putative binding site for NADH or substrate [60], was missed inthe/dhA gene (FIG. 3 c ).

Strain CTLABG1 was constructed via deletion of an isozyme (gpd1Δ) ofglyceraldehyde-3-phosphate dehydrogenase (Gpd) in the CTLABM strain.Donor DNA containing the homologous region of the upstream anddownstream of the GPD1 gene was synthesized using the gBlocks servicefrom Integrated DNA Technologies (IDT, IA, USA). The donor DNA wasamplified from the synthesized DNA using primer sets of dDNA-GPD1-F anddDNA-GPD1-R and integrated into the GPD1 locus of the CTLABM strainusing CRISPR-Cas9 based genetic modification, followed by selection onYPDNK plate. Colonies were randomly picked from the plate and verifiedby PCR amplification using primers (Conf-GPD1-F and Conf-GPD1-R).

Strain CTLABG2 was constructed via deletion of an isozyme (gpd2Δ) ofGpd. Donor DNA containing the homologous region of the upstream anddownstream of the GPD2 gene was synthesized using the gBlocks servicefrom Integrated DNA Technologies (IDT, IA, USA). The donor DNA wasamplified from the synthesized DNA using primer sets of dDNA-GPD2-F anddDNA-GPD2-R and integrated into the GPD2 locus of the CTLABM strainusing CRISPR-Cas9 based genetic modification as previously reported,followed by selection on YPDNH plate. Colonies were randomly picked fromthe plate and verified by PCR amplification using primers (Conf-GPD2-Fand Conf-GPD2-R).

Strain CTLABG1G2 was constructed via deletion of two isozymes (gpd1Δ,gpd2Δ) of Gpd in the CTLABM strain. The amplified donor DNAs for Gpd1deletion and Gpd2 deletion was integrated into the GPD1 locus and theGPD2 locus, respectively, followed by selection on YPDNHK plate.Colonies were randomly picked from the plate and verified by PCRamplification using primers (Conf-GPD1-F and Conf-GPD1-R, andConf-GPD2-F and Conf-GPD2-R), respectively.

Strain CBM was constructed via chromosomal integration of one copy ofcassette expressing tMDH3 gene. The tMDH3 expression cassette(P_(TDH3)-tMDH3-T_(CYC1)) was amplified from the pRS423_tMDH3 plasmidusing primer sets of dDNA-CS9-F and dDNA-CS9-R and integrated into theintergenic site on chromosome VIII (CS9) of the CTLABG1 G2 strain usingCRISPR-Cas9 based genetic modification, followed by selection on YPDNKplate. Colonies were randomly picked from the plate and verified by PCRamplification using primers (Conf-CS9-F and Conf-CS9-R).

Strain CBMM was constructed via the replacement of the/dhA gene with aheterologous ME1 gene from Rhodosporidium toruloides. The ME1 gene wascodon-optimized for S. cerevisiae and synthesized using the gBlocksservice from Integrated DNA Technologies (IDT, IA, USA). The synthesizedME1 gene was amplified using primer sets of dDNA-ME-F and dDNA-ME-R andintegrated into the/dhA gene in the PDC1 locus of the CBM strain usingCRISPR-Cas9 based genetic modification as previously reported, followedby selection on YPDNH plate. Colonies were randomly picked from theplate and verified by PCR amplification using primers (Conf-ME-F andConf-ME-R).

Strain CBMMP1 was constructed via the replacement of the endogenous PYC1promoter with a TEF1 promoter. The TEF1 promoter was amplified from thepRS426TEF plasmid using primer sets of dDNA-PYC1-F and dDNA-PYC1-R andintegrated into the PYC1 promoter region in the CBMM strain usingCRISPR-Cas9 based genetic modification as previously reported, followedby selection on YPDNK plate. Colonies were randomly picked from theplate and verified by PCR amplification using primers (Conf-PYC1-F andConf-PYC1-R).

Strain CBMMP2 was constructed via the replacement of the endogenous PYC2promoter with a PGK1 promoter. The PGK1 promoter was amplified from theplTy3PGK plasmid using primer sets of dDNA-PYC2-F and dDNA-PYC2-R andintegrated into the PYC2 promoter region in the CBMM strain usingCRISPR-Cas9 based genetic modification as previously reported, followedby selection on YPDNH plate. Colonies were randomly picked from theplate and verified by PCR amplification using primers (Conf-PYC2-F andConf-PYC2-R).

Strain CBMMP1P2 was constructed via the replacement of the endogenousPYC2 promoter with the PGK1 promoter in the CBMMP1 strain. The amplifiedPGK1 promoter was integrated into the PYC2 promoter region in the CBMMP1strain using CRISPR-Cas9 based genetic modification as previouslyreported, followed by selection on YPDNH plate. Colonies were randomlypicked from the plate and verified by PCR amplification using primers(Conf-PYC2-F and Conf-PYC2-R).

Example 2 Fermentation Experiments

To produce 2,3-BDO, we precultured engineered yeast strains overnight in5 mL of YPD20 (YP medium with 20 g/L of glucose) at 30° C. and 250 rpm.The precultured yeast cells were transferred into 50 mL of YPD20 andincubated under the same conditions. Grown cells were harvested at themid-exponential phase and inoculated into 25 mL of either YPD40 (YPmedium with 40 g/L of glucose) or YPD100 (YP medium with 100 g/L ofglucose) in a 125-mL flask with an initial cell density of OD₆₀₀=1.Either 100 rpm or 250 rpm of agitation was provided for oxygen-limitedor aerobic conditions, respectively.

Glucose fed-batch fermentation was conducted in a BioFlo & CelliGen 115fermenter (New Brunswick Scientific-Eppendorf, CT, USA) using a YPmedium. The S. cerevisiae CBMMP1P2 strain—the best strain identifiedfrom shake flask fermentation—was precultured in 200 mL of YPD100, theninoculated into 1L of YPD100 medium. After the initially added glucosewas depleted, additional glucose was added to the bioreactor up to 100g/L of glucose. The pH of fed-batch fermentation was controlled at pH5.6 by adding 2N NaOH.

Cell density measured at 600 nm (OD₆₀₀) was monitored using aspectrophotometer (BioMate 5, Thermo Fisher Scientific, MA, USA).Extracellular glucose, glycerol, ethanol, acetoin and 2,3-BDOconcentrations of culture broths were analyzed with the Agilent 1200HPLC system equipped with a refractive index detector (AgilentTechnologies, DE, USA) and Rezex ROA-Organic Acid H+(8%) column(Phenomenex, CA, USA). The flow rate of the mobile phase 0.005N H2SO4was 0.6 mL/min, and the column temperature was 50° C. To measurecellular concentrations of redox cofactor NADPH and NADP⁺, cells wereharvested by centrifugation at 21,130×g and 4° C. for 10 min. Cellpellets were washed using ice cold distilled water. NADPH and NADP⁺ wereextracted from yeast cells and determined by EnzyChrom NADP⁺/NADPH Assaykit (BioAssay Systems, CA, USA) and Synergy 2 microplate reader (BioTek,VT, USA).

Example 3 Techno-Economic Analysis (TEA) and Life Cycle Assessment (LCA)

To further investigate the potential of the CBMMP1P2 strain inindustrial application, we designed, simulated, and evaluatedlignocellulosic biorefineries producing MEK-a widely used industrialchemical (e.g., as a solvent, in coatings) [37]-via 2,3-BDO. We assumedthe CBMMP1P2 strain exclusively produced (2R,3R)-BDO enantiomer in thisstudy because the bacterial 2,3-BDO biosynthetic enzymes introduced inthe strain have been optimized to produce (R)-acetoin [38, 39]. Thus, weevaluated biorefinery economics and environmental impact based on theassumption of (2R,3R)-BDO production by the CBMMP1P2 strain.

Briefly, incoming lignocellulosic biomass is pretreated with dilutesulfuric acid to hydrolyze most of the xylan and a small amount ofglucan into xylose and glucose, respectively. The pretreated slurry issent to enzymatic hydrolysis where most of the glucan is converted toglucose. The hydrolysate is then concentrated to increase the sugarconcentrations and to remove fermentation inhibitors including aceticacid, furfural, and hydroxymethyl furfural (HMF). The concentratedhydrolysate is used as the substrate for continuous fermentation(overall 2,3-BDO yield) calculated assuming yield on xylose is 80% ofthat demonstrated on glucose to produce crude 2,3-BDO (the fermentationbroth), following which a series of solvent extraction and vacuumdistillation unit operations is used to separate and purify 2,3-BDO toapproximately 100 wt %. The purified 2,3-BDO is subsequently convertedto MEK (major) and isobutyraldehyde (IBA, minor) as products viacatalytic dehydration [6, 11, 40]. Finally, MEK is purified throughvacuum distillation to >99.5 wt % and sold as the main product of thebiorefinery, and IBA is hydrogenated into isobutyl alcohol (IBO), whichis purified to >99.5 wt % through distillation and sold as a co-product.

Details of Biorefinery Design and Analysis

Biorefinery Design

The biorefinery consists of five main processes: pretreatment,conversion (enzymatic hydrolysis, hydrolysate concentration, andfermentation), separation and upgrading, wastewater treatment, andfacilities (utilities and storage). Design of the pretreatment process,the enzymatic hydrolysis unit (including a holding tank), hydrolysateconcentration (through a multi-effect evaporator), the wastewatertreatment process, and facilities followed the same assumptions asdescribed in Bhagwat et al. [61].

Hydrolysate concentration is realized through a multi-effect evaporator(FIG. 13 ; full process flowsheet available online [62], which isincluded (i) to enable the evaluation of the biorefinery across a widerange of 2,3-BDO titer and yield combinations, as well as (ii) to reducethe maximum concentration of fermentation inhibitors (acetic acid,furfural, and hydroxymethyl furfural, all of which are volatile) tobelow 1 g/L [61]. Specifically, the hydrolysate is concentrated so thatat the target titer, the steady state concentration of 2,3-BDO in themain fermenter equals the target titer. In the case that not enoughinhibitors have been removed, the hydrolysate is concentrated to agreater level than needed for the target titer (so that more inhibitorscan be removed), and dilution water is added in the fermenter tomaintain the target titer. Further, a maximum sugar (glucose, xylose,mannose, arabinose, and galactose) concentration of 600 g/L was chosenas a constraint so that the viscosity of the concentrated hydrolysatewill not exceed the capacity of the centrifuge pump [61]. If atiter-yield combination requires a >600 g/L sugar concentration in theconcentrated hydrolysate (i.e., a high-titer, low-yield combination),the concentration is determined to be infeasible (blank areas in thetop-left portions of FIG. 8 a, 8 b, 8 c ).

For the fermentation units (a main fermenter and a seed train), the samedesign algorithms as in Bhagwat et al. [61] are used to size thereactors (based on the retention time calculated through target titerand productivity) and calculate the capital and operating costs, but thefermentation reactions are based on the experimental data obtained fromthe CBMMP1P2 strain. For the baseline analysis, a 2,3-BDO titer of 109.9g/L, a productivity of 1.0 g/L/h, and a yield of 0.36 g/g glucose wasused. It is assumed that 6.5% of the glucose and xylose is converted toacetoin (based on the terminal concentration from the fed-batchexperiment), and 2% is converted to cell mass [63]. As existing studieshave shown the ability of S. cerevisiae to utilize xylose [64], yield of2,3-BDO on xylose is assumed to be 80% of the corresponding yield onglucose (i.e., a yield of 80%×0.36 g/g glucose=0.288 g/g xylose). Basedon the composition of the saccharified slurry (mass of glucose: mass ofxylose=1.778:1), this corresponds to an overall 2,3-BDO fermentationyield of 0.334 g/g. Additionally, a 90% acetoin-to-2,3-BDO conversion(mol/mol) is assumed (acetoin removed from the downstream separationprocess is recycled to the fermentation broth to increase 2,3-BDOproduction). For the seed train, yields of all products are set to 80%of the main fermenter, but the acetoin-to-2,3-BDO conversion is excludedas no acetoin is recycled to the seed train.

The recovery of 2,3-BDO from the fermentation broth is challenging dueto both its low concentration and high solubility in water. Harvianto etal. [65] found that a hybrid extraction-distillation process was moreeconomical than conventional distillation for recovering 2,3-BDO andidentified oleyl alcohol as the best solvent out of six alternatives dueto its high selectivity. This study follows the proposed design usinghybrid extraction-distillation process with an oleyl alcohol solventwith a few minor modifications (FIG. 13 ; full process flowsheetavailable online [62]). The fermentation effluent is first distilled to250 g/L 2,3-BDO for a consistent recovery of 2,3-BDO across fermentationtiters. The total mass flow rate of solvent sent to the multi-stagemixer-settlers is 1.2 times the mass flow rate of water in the feed. Theextract is distilled at a pressure of 0.06 atm to operate the reboilerat a reasonable temperature (524.77 K) at which heating agents areavailable and to lower the heating requirement. The water, 2,3-BDO, andacetoin are then distilled by sequential conventional distillation at0.2 atm. The recovered acetoin sent back to fermentation to increase theoverall conversion to 2,3-BDO.

The purified (around 99.7 wt %) 2,3-BDO stream is then sent to catalyticdehydration at 300° C. with tricalcium phosphate as the catalyst. Duringthe reaction, 81% of the 2,3-BDO is assumed to be converted to MEK and9% to isobutyraldehyde (IBA) [66]. After the reaction, the reactantstream is passed through a distillation column with IBA separated as thetop product and MEK and 2,3-BDO in the bottom product. IBA is thenhydrogenated to isobutyl alcohol (IBO) using a kieselguhr-supportedcarboxymethylcellulose-nickel catalyst at 163° C. with an 86% molarconversion [67], and a >99.5 wt % pure IBO product can be obtained byremoving the unreacted IBA using another distillation column, and theremoved IBA is recycled back to the hydrogenation reactor. Finally, thebottom product containing MEK and unreacted 2,3-BDO is sent to anotherdistillation column, where a MEK product of >99.9 wt % purity can beobtained through vacuum distillation, and the unreacted 2,3-BDO isrecycled back to the dehydration reactor.

Design, simulation, TEA, and LCA of the biorefinery were performed usingthe open-source software BioSTEAM [23, 24], and the biorefinery designcan be accessed in the online repository [41]. Minimum product sellingprice (MPSP) was estimated by TEA (assuming a 10% internal rate ofreturn), and environmental impacts-namely, 100-year global warmingpotential (GWP₁₀₀) and fossil energy consumption (FEC)-were estimated bycradle-to-grave LCA that followed system boundary assumptions consistentwith the biorefinery LCA performed in Bhagwat et al. [42]. A systemreport is available in the online repository [41], containing a systemdiagram and information on the biorefinery mass and energy flows,capital and operating costs, and unit design requirements.

Techno-Economic Analysis and Life Cycle Assessment (TEA/LCA)

TEA was performed using BioSTEAM's cashflow analysis to calculate theminimum product selling price (MPSP) of MEK assuming a fixed IBO sellingprice of $1.3/kg (see Table 10 for all baseline prices). All TEAassumptions (e.g., plant size and lifetime, internal rate of return)were consistent with Bhagwat et al. [61]. Detailed information onindividual equipment costs, chemical prices, and utility (heating,cooling, and electricity) usages can be found in Tables 7, 8, and 9, andin the online biorefinery module [62].

The scope of the LCA was set to be cradle-to-grave to include impacts ofthe lignocellulosic feedstock and product degradation [61]. To calculatethe total impacts, mass and energy flows of input chemicals, andnon-biogenic emission (simulated using BioSTEAM) were combined with lifecycle inventory and impact characterization factors obtained from GREET2020 [68] and ecoinvent 3.7.1 [69]. To be consistent with the U.S.renewable fuel standard (RFS) [70], impacts associated with biorefineryconstruction were excluded. The functional unit was set to be 1 kg ofMEK, and the system expansion method was used assuming the bio-based IBOproduct from the biorefinery can replace the current petroleum-based IBO(i.e., impacts associated petroleum-based IBO was included as credits tocalculate the impacts for MEK). Two impact indicators, 100-year globalwarming potential (GWP₁₀₀) and fossil energy consumption (FEC), werechosen due to their prominent usage in the literature and theirrelevance to policies and legislation [71,72].

TABLE 7 Capital expenditures for the baseline biorefinery. Notes Cost[MM$] ISBL^(a) installed equipment cost — 122.035 OSBL^(b) installedequipment cost — 155.254 Warehouse 4.0% of ISBL 4.881 Site development9.0% of ISBL 10.983 Additional piping 4.5% of ISBL 5.492 Total directcost (TDC) — 298.645 Proratable costs 10.0% of TDC 29.864 Field expenses10.0% of TDC 29.864 Construction 20.0% of TDC 59.729 Contingency 30.0%of TDC 29.864 Other indirect costs 10.0% of TDC 29.864 (start-up,permits, etc.) Total indirect cost — 179.187 Fixed capital investment(FCI) — 477.832 Working capital 5.0% of FCI 23.892 Total capitalinvestment (TCI) — 501.723 ^(a)ISBL indicates inside-battery limits^(b)OSBL indicates outside-battery limits

TABLE 8 Variable operating costs for the baseline biorefinery. Price[$/ton] Cost [MM$ · yr⁻¹] Raw materials Enzyme 5588.260 27.932 Caustic136.078 1.600 Lime 180.870 0.263 Boiler chemicals 4532.171 1.644 Coolingtower chemicals 2721.555 0.160 Feedstock 57.008 51.624 Makeup water0.230 1.217 H₂ 934.401 0.263 By-products and credits Isobutanol 1179.34112.246 Ash disposal −28.860 −0.051 Raw materials Natural gas 197.76617.015 Total variable operating 89.523 cost

TABLE 9 Fixed operating costs for the baseline biorefinery. BaselineFermentation Performance Scenario Cost Notes [MM$ · yr⁻¹] Labor salary —3.213 Labor burden 90% of labor salary 2.892 Maintenance 3.0% of ISBL3.661 Property insurance 3.0% of ISBL 0.854

TABLE 10 List of parameters included in uncertainty analyses. ParameterUnit Baseline Distributions References Feedstock^(a) kg CO₂- 0.1090.096, 0.129, baseline from GREET [68], range from GWP₁₀₀ eq/dry-kguniform Kaliyan et al. [76] Feedstock^(a) FEC MJ/dry-kg 1.68 1.33, 1.76,baseline from GREET [68], range from uniform Kaliyan et al. [76] Plantuptime % of year 90% 84%, 90%, 96%, baseline and upper bound from Davistriangular et al. [63], lower bound set to be consistent with upperbound Total capital % of 100%  simulated scripts), range consistent withDavis et investment simulated value ± 25%, simulated by BioSTEAM(references value triangular for individual equipment noted in al. [63]Feedstock price $/dry-ton 71.3 60, 71.3, 83.7, baseline and lower boundfrom Davis triangular et al. [63], upper bound from Roni et al. [77]Sulfuric acid price $/kg 0.0948 0.0910, 0.0948, baseline from Davis etal. [63], range 0.1046, calculated using price index of sulfurictriangular acid during 2015-2019 from U.S. Bureau of Labor Statistics[78] Quicklime price $/kg 0.262 0.160, 0.262, baseline from Davis et al.[63], lower 0.288, triangular bound from National Minerals InformationCenter [79], upper bound is baseline + 10% Natural gas price $/kg 0.2530.198, 0.253, range is the minimum, average, and 0.304, triangularmaximum price during 2010-2019 [80] Oleyl alcohol $/kg 2.97 baseline ±10%, baseline adjusted for inflation from price triangular pricereported for market for fatty alcohol, GLO, ecoinvent 3.7.1 [69]Tricalcium $/metric 850 baseline ± 10%, baseline from vendor cited inthe phosphate tonne triangular online biorefinery module [62] catalystprice Kieselguhr- $/kg 4.80 baseline ± 10%, baseline estimated fromprices of supported triangular individual materials (based on thecarboxymethylcel composition of the catalyst described lulose-nickel byZhou et al. [67]; vendor cited in the catalyst online biorefinery module[62] H₂ price $/kg 1.03 baseline ± 10%, baseline from vendor cited inthe triangular online biorefinery module [62] Ash disposal^(b) $/kg−0.041 baseline ± 10%, baseline from Davis et al. [63] triangular Enzymeprice $/kg 6.16 baseline ± 10%, baseline from Li et al. [81] triangularGypsum $/kg 0 −0.0288, lower bound from National Minerals disposal^(b)0.00776, uniform Information Center [79], upper bound from Li et al.[81] Caustic materials $/kg 0.263 baseline ± 10%, baseline from Li etal. [81] is used for a for WWT price triangular 50 wt % mixture Make-upwater 10⁻² $/kg 0.044 baseline ± 10%, baseline from Li et al. [81] pricetriangular Electricity price $/kWh 0.070 0.067, 0.070, range is theminimum, average, and 0.074, triangular maximum price during 2010-2019(U.S. Energy Information Administration n.d.) Pretreatment mass % 30%25, 30%, 40%, baseline and range from Humbird et solids loadingtriangular al. [82] Pretreatment mg/g-dry 22.1 10, 22.1, 35, baselineand range from Humbird et sulfuric acid feedstock triangular al. [82]loading Pretreatment % of glucan 9.9%  6%, 9.9%, 12%, baseline and rangefrom Humbird et glucan-to-glucose triangular al. [82] conversionPretreatment % of xylan 90% 80%, 90%, 92%, baseline and range fromHumbird et xylan-to-xylose triangular al. [82] conversionSaccharification mass % 20% 17.5%, 20%, baseline and range from Humbirdet solids loading 25%, triangular al. [82] Saccharification Mg/g-glucan20 10, 20, 30, baseline and range from Humbird et enzyme loadingtriangular al. [82] Saccharification h 24 0, 24, 56, baseline based onHumbird et al. [82], time triangular range from Li et al. [81]Saccharification % of glucan 90% 75%, 90%, 95%, baseline and range fromHumbird et glucan-to-glucose triangular al. [82] conversion CSL loadingg/L 10 5, 10, 15, baseline and range based on Li et al. triangular [81]2,3-BDO yield g/g 49% 0.334 ± 20%, baseline is 0.36 (for glucose) and0.3 triangular from Table 4 2,3-BDO titer g/L 54.8 achieved value ±baseline from Table 4 20%, triangular 2,3-BDO g · L⁻¹ · h⁻¹ 0.76achieved value ± baseline from Table 4 productivity 10%, triangularInoculum ratio %  7% 5%, 7%, 10%, baseline and range based on that oftriangular lactic acid [81] Acidulation time h 1 baseline ± 10%,baseline from BioSTEAM mix tank triangular Gypsum % 99.5%   95%, 99.5%,baseline based on Aden et al. [83] separation 100%, triangularDehydration mol % 90.0%   81.0%, 90.0%, baseline from Penner et al.[66], conversion 99.0%, triangular bounds are baseline ± 10% DehydrationMEK % 90.0%   81.3%, 90.0%, baseline from Penner et al. [66],selectivity 96.0%, upper bound from Emerson, triangular Flickinger, andTsao [84], lower bound from Zhao et al. [85] Dehydration IBA % 10.0%  9.0%, 10.0%, baseline from Penner et al. [66], selectivity 11.0%,triangular bounds are baseline ± 10% Dehydration time h 0.5 baseline ±10%, baseline from Emerson, Flickinger, triangular and Tsao [84]Dehydration C. 200 180, 200, 220, baseline from Penner et al. [66] andtemperature triangular Zhao et al. [85], lower bound from Emerson,Flickinger, and Tsao [84], upper bound is baseline + 10% Hydrogenationmol % 86.0%   77.4%, 86.0%, baseline from Zhou et al. [61], boundsconversion 94.6%, triangular are baseline ± 10% Hydrogenation h 16baseline ± 10%, baseline from Zhou et al. [61] time triangularHydrogenation C. 25 baseline ± 10%, baseline from Zhou et al. [61]temperature triangular Boiler efficiency % 80% baseline ± 10%, baselinefrom Humbird et al. [82], uniform Davis et al. [63] ^(a)Farming(excluding credit for fixed carbon), harvest & collection,transportation, storage, handling, and pre-processing. GREET consideredonly farming impacts; impacts for other processes from Kaliyan, Morey,and Tiffany were added to the GREET value. ^(b)A negative ash/gypsumdisposal price indicates it is sold as a co-product.

Uncertainty Analysis

To characterize the uncertainty in MPSP, GWP100, and FEC of the baselinebiorefinery (FIG. 7 ) and of the biorefineries operating at alternativeglycerol yields (FIG. 12 ), samples were generated from thedistributions described in Table 10 for each of the 42 fluctuatedparameters using Latin hypercube sampling to reduce the time required toyield reproducible results.

Example 4 Measurement of 2,3-BDO Induced Drought Tolerance inArabidopsis Plants

To examine the feasibility of yeast 2,3-BDO fermentation broth as abiostimulant for drought tolerance in Arabidopsis plants, without anycomplicated purification steps, we obtained the fermentation broth(109.9 g/L, 13.8 mM 2,3-BDO) by separating the yeast cells viacentrifugation (4,000×g, 30 min) and diluted the broth to contain 250 μM2,3-BDO. Arabidopsis thaliana Col-0 seeds were surface-sterilized for 5min in 95% (v/v) ethanol followed by 5 min in 50% (w/v) NaClO [34]. Theseeds were incubated in sterile water for 3 days in the dark at 4° C.after an extensive wash with sterile distilled water. The incubatedseeds were spread evenly on solid Murashige-Skoog medium [43] forgermination. After 14 days, the seedlings were transplanted to potscontaining sterile vermiculite with fertilizer and grown in a controlledgrowth chamber (8/16 h light/dark cycle, light intensity of 200 μE m⁻²s⁻¹, 23/18° C., and 50% relative humidity). After 14 days, the rootinoculation was performed by pipetting 5 ml of the yeast fermentationbroth containing 250 μM 2,3-BDO or 5 ml of distilled water as a controlinto the vermiculite in the pots containing the plants. After 7 days,the plants were exposed to drought stress for 26 days followed byrehydration for 2 days and seedling survival was determined. Threeindependent experiments were performed, with at least 17 plants per eachtreatment. The survival rate (SR) of A. thaliana under drought stresswas counted from three independent experiments and calculated using thefollowing equation: SR (%)=survived plants after drought/treated plantsbefore drought.

Example 5 Results and Discussion

Construction of a Non-Ethanologenic S. cerevisiae

Pdc⁻-based S. cerevisiae has been used as a platform strain forproducing non-ethanol products. However, the Pdc⁻ strain exhibits growthdefects on glucose medium, hindering its usage as a platform strain forindustrial production of various bio-based products. The impaired growthof the Pdc⁻ strain can be explained by two main reasons. First, the Pdc⁻strain cannot synthesize acetyl-CoA, a precursor of necessary componentsfor cell growth. Second, the Pdc⁻ strain exhibits severe growth defectson glucose due to redox imbalance.

We hypothesized that a non-ethanologenic strain capable of growing onglucose without growth defects could be constructed by modulating theexpression levels of Pdc and Adh (FIG. 1 ). To verify the hypothesis, weconstructed three engineered S. cerevisiae strains (CTL, CTLA, andCTLAP) with different genetic modifications of Pdc and Adh isozymes. TheCTL strain was constructed by deleting a major isozyme of Pdc (pdc1Δ) inthe S. cerevisiae CT2 strain and simultaneously introducing/dhA encodinglactate dehydrogenase (LDH) from Rhizopus oryzae into the PDC1chromosome. The CTLA strain was constructed by additionally deleting amajor isozyme of Adh (adh1A) in the CTL strain. The endogenous PDC5promoter in the CTLA strain was replaced with a relatively weak TEX1promoter, and the resulting strain was named CTLAP.

To investigate the effects of different expression levels of Pdc and Adhisozymes on ethanol production, batch fermentations were performed inYPD40 under oxygen-limited conditions (FIG. 2 ). The CTL strainexhibited a 30% reduction in ethanol yield (0.28 vs. 0.40 g/g) ascompared with the CT2 strain and produced lactate with a yield of 0.22g/g while maintaining a high glucose consumption rate (2.8 g/L/h). TheCTLA strain produced lactate with a yield of 0.40 g/g as a majorbyproduct and showed 67.5% and 39.3% reductions in ethanol yield (0.40vs. 0.13 g/g) and glucose consumption rate (2.8 vs. 1.7 g/L/h) ascompared with the CT2 strain, respectively. The lower glucoseconsumption rate of the CTLA strain might be explained by the fact thatlactate production decreased the pH of culture medium, negativelyaffecting cell growth. The CTLAP strain exhibited a 70% reduction inethanol production but could not consume glucose efficiently after 24hours, leading to a 96.4% reduction in glucose consumption rate (2.8 vs.0.1 g/L/h) as compared with the CT2 strain. This result suggests thatmodulating the expression of the PDC5 gene might result in a limitedsynthesis of cytosolic acetyl-CoA in the CTLAP strain because Pdc5 isalso a major isozyme of Pdc in S. cerevisiae [47]. Based on theseresults, the deletion of major isozymes of Pdc and Adh (pdc1A and adh1A)can effectively prevent the cell defects caused by eliminating all Pdcisozymes (pdc1Δ, pdc5A, pdc6A) while reducing ethanol production (FIG.10 ). Thus, we selected the CTLA strain as a host strain for furtherexperiments.

Introduction of a 2,3-BDO Pathway into the Non-Ethanologenic YeastStrain

To produce 2,3-BDO by engineered yeast, S. cerevisiae CTLAB—the CTLAstrain with chromosomal integration of alsS and alsD expressioncassettes—was cultured in YPD40 under oxygen-limited conditions (FIG. 3). The CTLAB strain produced 17.9 g/L of lactate as a major product witha yield of 0.40 g/g alongside 4.9 g/L of acetoin and 1.9 g/L of 2,3-BDO(FIG. 3 a ). Interestingly, the CTLAB strain did not produce ethanol ascarbon flux was redirected from ethanol to lactate, acetoin, and2,3-BDO. The redox imbalance caused by the deletion of Pdc1 and Adh1(pdc1A and adh1A) is seemingly resolved by producing lactate and2,3-BDO, which are NADH-dependent products, instead of ethanol.

However, the CTLAB strain produced glycerol instead of lactate as amajor product with 2,3-BDO in batch fermentation after curing ofCas9-NAT and guide RNA plasmids (FIG. 3 b ). Sequencing the/dhA gene inthe CTLAB strain revealed a missing 117th amino acid (valine), which isthe putative binding site for NADH or substrate [48](FIG. 3 c ). Themutant strain was named CTLABM and found to produce 13.7 g/L of 2,3-BDOwith a yield of 0.34 g/g and 12.1 g/L of glycerol at a yield of 0.30g/g. The glucose consumption and growth rates of the CTLABM strainincreased 1.8-fold (2.22 vs. 1.26 g/L/h) and 1.9-fold (OD₆₀₀: 7.1 vs.3.8), respectively, as compared to the CTLAB strain. We reasoned thatthe spontaneous mutation on the/dhA gene might help to avoid a negativegrowth effect from lactate production during fermentation. However, aconsiderable amount of glycerol (0.30 g/g glucose) was produced as abyproduct because 2,3-BDO production from glucose is not redox neutral[49]. To further improve 2,3-BDO production by engineered yeast, it isnecessary to eliminate glycerol accumulation.

Elimination of Glycerol Production

The glycerol biosynthetic pathway is comprised of NADH-dependentglycerol-3-phosphate dehydrogenase (Gpd) and glycerol-3-phosphatephosphatase (Gpp) (FIG. 1 ). Gpd is known to be more directly involvedin glycerol production, and S. cerevisiae has two Gpd isozymes, Gpd1 andGpd2. The Gpd isozymes serve distinct physiological roles attributed todifferent transcriptional regulations. Gpd1 is responsible for osmoticstress-induced glycerol production, whereas Gpd2 is involved in redoxregulation [50].

To investigate the effects of the deletion of Gpd isozymes on 2,3-BDOproduction, we constructed three engineered yeast strains (CTLABG1,CTLABG2, and CTLABG1 G2), and batch fermentations were performed inYPD40 under oxygen-limited conditions. The CTLABG1 and CTLABG2 strainswere constructed by single deletion of GPD1 or GPD2 gene (gpd1Δ orgpd2Δ) in the CTLABM strain, respectively, while the CTLABG1 G2 strainwas constructed by double deletion of GPD1 and GPD2 genes (gpd1Δ andgpd2Δ) in the CTLABM strain. The deletion of Gpd isozymes affectedglucose consumption rate, glycerol, ethanol, and 2,3-BDO production inthe engineered strains (FIG. 4 , FIG. 11 ). Single deletion of GPD1 orGPD2 gene resulted in 36.7% and 60.0% reduction in glycerol yields (0.19and 0.12 vs. 0.30 g/g), respectively, as compared to the CTLABM strain.As Gpd2 is involved in redox regulation, the deletion of GPD2 gene wasmore effective in reducing glycerol production than the deletion of GPD1gene. As expected, double deletion of GPD1 and GPD2 genes resulted inthe complete elimination of glycerol production. However, along with thereduction in glycerol production, ethanol was produced again as aby-product in the GPD deletion strains (CTLABG1, CTLABG2, and CTLABG1G2) (FIG. 4 a ). We reasoned that ethanol production is an inevitablecompensation mechanism to restore the redox imbalance caused by thedeletion of Gpd isozymes. The double GPD deletion strain (CTLABG1 G2)exhibited a lower glucose consumption rate (1.28 g/L/h) than the singleGPD deletion strains (2.29 g/L/h for CTLABG1 and 1.67 g/L/h for CTLABG2)and their parental strain (2.22 g/L/h for CTLABM) (FIG. 11 ). Thisreduced glucose consumption rate might result from the limited NADHreoxidation capability caused by the deletion of Gpd isozymes. Alongwith the reduction in glucose consumption rates, volumetric 2,3-BDOproductivities of the GPD deletion strains were also drasticallyreduced. The single GPD deletion strains (CTLABG1, CTLABG2) and thedouble GPD deletion strain (CTLABG1G2) showed 35.6%, 38.2%, and 44.8%reduction in volumetric 2,3-BDO productivity (0.49, 0.47, 0.42 vs 0.76g/L/h) as compared to the CTLABM strain (FIG. 4 b ). Although theCTLABG1 G2 strain exhibited a low 2,3-BDO productivity after eliminationof glycerol production, this phenotype would still provide advantages indownstream processing of 2,3-BDO. As such, we selected the CTLABG1 G2strain as a host strain for further modifications.

We hypothesized that oxygen supply for regenerating NAD⁺ is an importantfactor for improving 2,3-BDO productivity and reducing ethanolproduction in the CTLABG1G2 strain [16]. To evaluate the effects ofaeration on 2,3-BDO and ethanol productions, a batch fermentation wasperformed in YPD100 either under oxygen-limited or aerobic conditions(Table 6).

TABLE 6 Summarized results of batch fermentations by the CTLABG1G2strain under oxygen-limited or aerobic conditions in YPD100. GlucoseYield of 2,3- Products (g/L) consumption Yield of 2,3- BDO 2,3- rateEthanol BDO productivity Condition DCW BDO Glycerol Ethanol (g/L/h)(g/g) (g/g) (g/L/h) Oxygen- 6.1 ± 29.1 ± N.D^(a) 18.0 ± 1.94 ± 0.15 ±0.25 ± 0.48 ± limited 0.1 1.2 0.1 0.00 0.00 0.01 0.02 (100 rpm) Aerobic6.6 ± 35.0 ± N.D^(a) 15.0 ± 3.22 ± 0.13 ± 0.28 ± 0.97 ± condition 0.20.1 1.4 0.00 0.00 0.01 0.00 (250 rpm) ^(a)N.D indicates not detected.

As a result, the CTLABG1 G2 strain exhibited 1.1-, 2.0-, and 1.7-foldhigher 2,3-BDO yield (0.28 vs. 0.25 g/g), 2,3-BDO productivity (0.97 vs.0.48 g/L/h), and glucose consumption rate (3.22 vs. 1.94 g/L/h) underaerobic conditions than oxygen-limited conditions, respectively. Thisresult indicated that respiration might help to restore redox imbalancecaused by the elimination of Gpd isozymes (gpd1Δ and gpd2Δ) and therebyincreasing 2,3-BDO yield and productivity. However, the CTLABG1 G2strain still produced ethanol with a yield of 0.13 g/g as a byproducteven under aerobic conditions, suggesting NAD⁺ regenerating capabilityby respiration was insufficient to eliminate ethanol production. Assuch, the introduction of an efficient NAD⁺ regenerating supplementarypathway was required for further improvements in 2,3-BDO production.

Recovering Redox Imbalance by Employing Pyruvate-Malate (PM) Cycle

To minimize ethanol production via resolving the redox imbalance, weconstructed the CBMM strain by introducing an NAD⁺ regenerating PM cycleconsisting of pyruvate carboxylase (PYC1 and PYC2), malate dehydrogenase(MDH3), and heterologous malic enzyme (ME1) from Rhodosporidiumtoruloides into the CTLABG1 G2 strain (FIG. 5 a ) [21]. To expressmalate dehydrogenase in the cytosol, a truncated version of MDH3 (tMDH3)without a peroxisomal targeting sequence was overexpressed.

Through the PM cycle, 1 mole of NADH can be converted into 1 mole ofNADPH. We hypothesized that the PM cycle can re-oxidize excess NADH,which might help to reduce ethanol production in the CBMM strain. Todetermine the effects of the PM cycle on ethanol production, batchfermentation was performed in YPD100 under aerobic conditions. However,we could not observe the positive effects of the PM cycle with respectto ethanol and 2,3-BDO production in the CBMM strain (FIG. 5 b , Sc).Counterintuitively, the CBMM strain exhibited slightly higher ethanolyield (0.14 vs. 0.13 g/g glucose) and lower 2,3-BDO yield (0.25 vs. 0.28g/g glucose) and productivity (0.84 vs. 0.97 g/L/h) than those of theCTLABG1 G2 strain. We reasoned that the expression of PYC1 and PYC2genes might be inhibited by transcriptional regulation in the presenceof glucose because the pyruvate carboxylase (PYC) participates inreplenishing oxaloacetate for gluconeogenesis when glucose has beendepleted.

To enhance the expression levels of pyruvate carboxylase in the PMcycle, we constructed three engineered S. cerevisiae strains (CBMMP1,CBMMP2, and CBMMP1P2) by replacing endogenous PYC1 and PYC2 promoterswith strong constitutive promoters. The CBMMP1 strain expressed PYC1with a strong TEF1 promoter, while the CBMMP2 strain expressed PYC2 witha strong PGK1 promoter. Lastly, the CBMMP1P2 strain expressed PYC1 withthe TEF1 promoter and PYC2 with the PGK1 promoter [53]. We then examined2,3-BDO and ethanol production of the three engineered strains in YPD100under aerobic conditions.

When we replaced the PYC1 promoter with the TEF1 promoter, the CBMMP1strain showed a 35.8% reduction in ethanol yield (0.09 vs. 0.14 g/g) and27.3% and 32.0% increases in 2,3-BDO productivity (1.07 vs. 0.84 g/L/h)and yield (0.33 vs. 0.25 g/g), respectively, as compared to the CBMMstrain. When we replaced the PYC2 promoter with the PGK1 promoter, theCBMMP2 strain also exhibited a 28.6% reduction in ethanol yield (0.10vs. 0.14 g/g) and 4.76% and 12.0% increases in 2,3-BDO productivity(0.88 vs. 0.84 g/L/h) and yield (0.28 vs. 0.25 g/g), respectively, ascompared to the CBMM strain. When the PYC1 and the PYC2 promoters weresimultaneously replaced with the TEF1 and PGK1 promoters, respectively,the CBMMP1P2 strain showed a 71.5% reduction in ethanol yield (0.04±0.01vs 0.14±0.02 g/g glucose) and 21.4% and 36.0% increases in 2,3-BDOproductivity (1.02±0.06 vs. 0.84±0.07 g/L/h) and yield (0.34±0.02 vs.0.25±0.02 g/g), respectively, as compared to the CBMM strain. Theseresults suggest that the overexpression of PYC1 and PYC2 genes viareplacement of its endogenous promoters might enable re-oxidizing excessNADH caused by the elimination of Gpd isozymes, resulting in a 71.5%reduction in ethanol production in the CBMMP1P2 strain compared with theCBMM strain.

To verify the enhancement of NAD⁺ regenerating capability of the PMcycle via overexpressing the PYC1 and PYC2 genes, we measured thecellular concentrations of redox cofactors NADPH and NADP⁺ in theengineered yeast strains (CBMM and CBMMP1P2) (FIG. 5 d ). As a result,the intracellular ratio of NADPH/NADP⁺ in the CBMMP1P2 strain was 34%higher than in the CBMM strain, indicating that the NAD⁺ regeneratingcapability of the PM cycle was enhanced in the CBMMP1P2 strain. As such,we envision that the PM cycle can be a promising NAD⁺ regeneratingsupplementary pathway for efficient 2,3-BDO production by engineeredyeast.

Fed-Batch Fermentation for 2,3-Butanediol Production

To increase the titer of 2,3-BDO and to investigate the feasibility oflarge-scale production of 2,3-BDO, the performance of the CBMMP1P2strain—the best strain identified from shake flask fermentation—wastested in a bioreactor while feeding glucose to maximize 2,3-BDOproduction (FIG. 6 ). In fed-batch fermentation, 304.7 g/L of glucosewas consumed and 109.9 g/L of 2,3-BDO was produced with a productivityof 1.0 g/L/h and a yield of 0.36 g/g glucose (FIG. 6 ). Glycerol andethanol accumulation were not observed.

In the feeding II phase, the CBMMP1P2 strain exhibited 52.2% and 47.7%increases in glucose consumption rate (3.5 vs. 2.3 g/L/h) and 2,3-BDOproductivity (1.30 vs. 0.88 g/L/h) than those of the feeding I phase.This result suggested that the CBMMP1P2 strain mainly utilized glucosefor cell growth rather than 2,3-BDO production in the feeding I phasedue to the low initial cell density. We expected that the glucoseconsumption rate and 2,3-BDO productivity might be improved in thefeeding I phase if high cell density was inoculated at the beginning offed-batch fermentation. In the feeding III phase, the CBMMP1P2 strainshowed the highest 2,3-BDO yield (0.45 g/g) among the feeding phases(feeding I: 0.35, feeding II: 0.37 g/g) of the fed-batch fermentationwith a 2,3-BDO productivity of 1.30 g/L/h.

Although we obtained promising results in producing 2,3-BDO throughrational metabolic engineering efforts, further improvements to theCBMMP1P2 strain can be considered. About 10 g/L of acetoin, the oxidizedform of 2,3-BDO, was accumulated during the fed-batch fermentation. Wespeculated that the insufficient activity of endogenous butanedioldehydrogenase (Bdh1) due to the cofactor imbalance might causeaccumulation of acetoin. Because of the NAD⁺ regenerating capability ofPM cycle (FIG. 5 a, 5 d ), the intracellular ratio of NADPH/NADH mightbe increased in the CBMMP1P2 strain. As the Bdh1 converts acetoin into2,3-BDO using NADH as a cofactor, the increased intracellular NADPH/NADHratio might be disadvantageous for an efficient Bdh1 enzymatic reaction.As a possible solution for this limitation, the additional expression ofheterologous butanediol dehydrogenase using NADPH as a cofactor may helpto reduce the acetoin accumulation and further improve the 2,3-BDO yieldand productivity.

Implications on Biorefinery Sustainability

Under the baseline fermentation performance assumptions demonstrated bythe CBMMP1P2 strain (titer of 109.9 g/L, overall yield of 0.334 g/g onglucose and xylose, and productivity of 1.0 g/L/h) in this study, thebiorefinery can produce MEK at an MPSP of $1.90/kg (baseline) with arange of $1.66-2.27/kg [5t^(h)-95t^(h) percentiles from the uncertaintyanalysis, hereafter shown in brackets]. The baseline MPSP is within themarket price range ($1.40-1.98/kg; FIG. 7 a ), and the MPSP is at orbelow the high end ($1.98/kg) of the market price range in 61.2% ofsimulations in the uncertainty analysis. If, instead of upgrading2,3-BDO to MEK and IBO, the biorefinery's filtered fermentation brothwere sold directly at 109.9 g/L (13.8 mM 2,3-BDO)-which may be dilutedby the user to 250 μM 2,3-BDO and utilized to induce drought toleranceas described in section 2.6)—the MPSP of the 2,3-BDO contained in thebroth (under assumptions otherwise consistent with the baseline case)would be $2.52/kg 2,3-BDO [$2.35-2.81/kg 2,3-BDO]assuming a 7-daystorage time for the high-volume broth or $1.25/kg 2,3-BDO[$1.09-1.53/kg 2,3-BDO] neglecting product storage costs (i.e., assumingimmediate sale).

Additionally, future advancements in fermentation yield would enhancethe financial viability of the MEK biorefinery (FIG. 8 a ). For example,with a modest 14.5% increase in the overall yield (from 0.334 to 0.382g/g), the MPSP of MEK would decrease to match the mid-point of themarket price range at $1.69/kg. Moreover, the fermentation performanceobserved during feeding phase III of the fed-batch experiment wassignificantly better than the overall fed-batch performance (FIG. 6 ).It may be possible to reproduce the conditions responsible for improvedfermentation performance (e.g., 2,3-BDO productivity and yield) on anindustrial scale via continuous fermentation. If the fermentationperformance observed in feeding phase III of the fed-batch experimentwere assumed (titer of 109.9 g/L, overall yield of 0.418 g/g on glucoseand xylose, and productivity of 1.3 g/L/h), MPSP of MEK would bedecreased to $1.57/kg [$1.40-1.88/kg], which is consistently (in 98.8%of simulations) within or below the market price range.

With regard to life cycle environmental impacts, GWP₁₀₀ and FEC of MEKunder baseline assumptions were found to be 0.37 kg CO₂ eq/kg[-0.46-1.53 kg CO₂ eq/kg] and 3.1 MJ/kg [−6.9-19.8 MJ/kg], respectively,which were consistently lower than those reported for petroleum-basedMEK (3.48-4.36 kg CO₂ eq/kg and 43.0-58.7 MJ/kg, respectively [55, 56];FIG. 7 b, 7 c ). GWP₁₀₀ and FEC were below zero in 17.9% and 23.1% ofthe simulations, respectively, due to the sale of electricityoverproduced by the turbogenerator (which was assumed to displace gridelectricity). Notably, unlike MPSP, which was influenced mainly by yield(FIG. 8 a ), environmental impacts of the produced MEK were largelytiter-driven (FIG. 8 b, 8 c ). This is mainly caused by the separationand upgrading process, where seven distillation columns are used,resulting in a high contribution to the system heating demand (FIG. 7 a). Therefore, a higher titer can further enhance the sustainability ofthe biorefinery, with GWP₁₀₀ and FEC decreasing from 0.37 kg CO₂ eq/kgand 3.1 MJ/kg to −0.86 kg CO₂ eq/kg and −12.5 MJ/kg, respectively, whentiter is increased from the baseline of 109.9 g/L to 178 g/L (thehighest 2,3-BDO titer using S. cerevisiae reported in the literature[18]) along with a modest yield improvement to 0.402 g/g (for a feasibletiter-yield combination; FIG. 8 b, 8 c ). As a high productivity wasmaintained even at the high titers toward the end of the fed-batchexperiment (FIG. 6 ), the CBMMP1P2 strain may be able to achieve highertiters (than those obtained in this study) in continuous fermentation,when operating in a steady state mode.

As glycerol presence hindered the liquid-liquid equilibrium duringsolvent extraction, the decreased glycerol yields are beneficial to thefinancial viability of the biorefinery. Increased glycerolconcentrations during solvent extraction resulted in decreased partitionof 2,3-BDO into the solvent (necessitating greater amounts of solventfor equal 2,3-BDO recovery) and increased fractional loss of solvent inthe raffinate. This incurred greater costs from purchase of make-upsolvent and higher utility requirements for downstream separation. Forexample, increasing glycerol yield from 0.00 g/g to 0.20 g/g wouldresult in an increase in MPSP from $1.90/kg [$1.66-2.27/kg] to $2.84/kg[$2.56-3.18/kg] under the baseline fermentation performance (FIG. 12 ),highlighting the contribution of this work to decrease glycerolproduction during 2,3-BDO production. Overall, as supported by the TEAand LCA results, the CBMMP1P2 strain is highly promising for sustainableMEK and IBO production via catalytic dehydration of 2,3-BDO fromlignocellulosic feedstocks.

Usage of 2,3-BDO as a Biostimulant

Evidence from many studies indicates that 2,3-BDO and acetoin modulate anetwork of metabolic events involved in triggering hormonal responsesaimed at protecting plants [25-28, 54]. Particularly, (2R,3R)-BDO hasbeen reported to be effective for drought tolerance in plants [33, 34].Cho et al. [33] showed that (2R,3R)-BDO released by Pseudomonaschlororaphis strain 06 induced systemic drought tolerance in Arabidopsisthaliana, and this trait is associated with the increased accumulationof salicylic acid (SA), modulating stomatal closure. Wu et al. [34] alsoreported that root colonization with Bacillus amyloliquefaciens ordirect root treatment with 250 μM of acetoin and 2,3-BDO triggered theSA signaling networks and stimulated the production of hydrogen peroxide(H₂O₂) and nitric oxide (NO), inducing stomatal closure in Arabidopsisthaliana and Nicotiana benthamiana (tobacco).

We hypothesized that as the yeast 2,3-BDO fermentation broth obtained inthis study does not contain by-products such as glycerol and ethanol,drought tolerance can be induced even when treated with plants withoutany complicated purification process. To test the feasibility of theyeast 2,3-BDO fermentation broth as a biostimulant for inducing droughttolerance in plants, we inoculated the roots of growth-chamber grown A.thaliana plants with fermentation broth or distilled water as a control(FIG. 9 ). After 26 days of drought stress, the survival rate (SR) offermentation broth-treated plants was 3.8-fold higher than the distilledwater-treated plants (41.4% vs. 10.8%). This result suggested that yeast2,3-BDO fermentation broth could induce drought tolerance in Arabidopsisplants and be used as a biostimulant for plant health in the agricultureindustry.

Example 6 Conclusion

This study combined experimental yeast 2,3-BDO production fromlignocellulosic biomass with two potential downstream uses: (i) as afeedstock for MEK production via catalytic dehydration of 2,3-BDO and(ii) as a biostimulant to induce drought tolerance in plants. Ourengineered yeast strain produced 109.9 g/L of 2,3-BDO with aproductivity of 1.0 g/L/h without ethanol and glycerol in fed-batchfermentation through metabolic reprogramming. When a TEA was conductedbased on the experimental results, the MPSP ($1.90/kg [$1.66-2.27/kg])of produced MEK was within the market price range ($1.40-1.98/kg) ofpetroleum-based MEK. Regarding cradle-to-grave LCA, both GWP₁₀₀ (0.37 kgCO₂ eq/kg [-0.46-1.53 kg CO₂ eq/kg]) and FEC (3.1 MJ/kg [−6.9-19.8MJ/kg]) impacts of the produced MEK were significantly lower than thepreviously reported values (3.48-4.36 kg CO₂ eq/kg and 43.0-58.7 MJ/kg,respectively) for petroleum-based MEK. This study demonstrated thepotential for economical and sustainable bio-based MEK production fromlignocellulosic biomass. In addition, as another potential applicationof 2,3-BDO, we demonstrated that yeast 2,3-BDO fermentation broth couldbe used as a biostimulant for inducing drought tolerance in Arabidopsisplants without a complicated purification process.

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1. A recombinant yeast cell comprising: (a) a genetic modification toreduce or eliminate expression of glyceraldehyde-3-phosphatedehydrogenase encoded by GPD1 and GPD2; (b) a genetic modification toreduce or eliminate expression of pyruvate decarboxylase encoded byPDC1; (c) a genetic modification to reduce or eliminate expression ofalcohol dehydrogenase encoded by ADH1; (d) a heterologous nucleic acidmolecule encoding acetolactate synthase (alsS); (e) a heterologousnucleic acid molecule encoding acetolactate decarboxylase (alsD); (f) agenetic modification to increase expression of pyruvate decarboxylaseencoded by PYC1 and PYC2 as compared to expression of pyruvatedecarboxylase in a wild-type yeast cell; (g) a heterologous nucleic acidmolecule encoding malate dehydrogenase (Mdh3); and (h) a heterologousnucleic acid molecule encoding malic enzyme (Me1).
 2. The recombinantyeast cell of claim 1, further comprising: (i) a heterologous nucleicacid molecule encoding xylose reductase (Xyl1); (j) a heterologousnucleic acid molecule encoding xylitol dehydrogenase (Xyl2); (k) aheterologous nucleic acid molecule encoding xylulokinase (Xyl3); (I) agenetic modification to reduce or eliminate expression of4-nitrophenylphosphatase (Pho13); and (m) a genetic modification toreduce or eliminate expression cytosolic aldehyde dehydrogenase (Ald6).3. The recombinant yeast cell of claim 1, further comprising aheterologous nucleic acid molecule encoding butanediol dehydrogenase(Bdh1).
 4. (canceled)
 5. (canceled)
 6. The recombinant yeast cell ofclaim 1, wherein the genetic modification to increase expression ofpyruvate decarboxylase encoded by PYC1 and PYC2 comprises a strongpromoter operably linked to the PYC1 and PYC2.
 7. The recombinant yeastcell of claim 6, wherein the strong promoter is a TEF1 promoter or aPGK1 promoter.
 8. (canceled)
 9. The recombinant yeast cell of claim 1,wherein the nucleic acid molecule encoding Mdh3 encodes a truncated Mdh3(tMdh3), wherein the last three amino acids (SKL) are absent.
 10. Therecombinant yeast cell claim 8, wherein the nucleic acid moleculeencoding Mdh3 is operably linked to a strong promoter.
 11. (canceled)12. (canceled)
 13. (canceled)
 14. The recombinant yeast cell of claim 1,wherein the recombinant yeast cell can ferment xylose.
 15. A yeast cellculture comprising two or more of the recombinant yeast cells ofclaim
 1. 16. A method of producing 2,3-butanediol (2,3-BDO) comprisingcontacting a substrate with the recombinant yeast cell of claim
 1. 17.The method of claim 16, wherein the substrate is lignocellulosic orcellulosic feedstock.
 18. (canceled)
 19. The method of claim 16, whereinsubstantially no glycerol or ethanol is accumulated.
 20. The method ofclaim 16, wherein less than 2 g/L of ethanol and less than 2 g/L ofglycerol is accumulated.
 21. The method of claim 16, wherein 2,3-BDO isproduced at more than 0.5 g/L/h or at more than 1.0 g/L/h.
 22. Themethod of claim 16, wherein 2,3-BDO is produced at a yield of 100 g/L ormore.
 23. A method of producing methyl ethyl ketone (MEK) comprising:(a) contacting a substrate with the recombinant yeast cell of claim 1under fermentation conditions; (b) collecting and purifying 2,3-BDO toform purified 2,3-BDO; (c) subjecting the purified 2,3-BDO to catalyticdehydration such that MEK is produced.
 24. The method of claim 23,wherein, a catalyst for the catalytic dehydration is tricalciumphosphate.
 25. The method of claim 23, wherein, the purified 2,3-BDO isgreater than 90 wt % pure.
 26. A fermentation broth produced bycontacting the recombinant yeast cell of claim 1 with a fermentationmedium.
 27. A method of inducing drought tolerance in plants comprisingcontacting roots of the plants with the fermentation broth of claim 26.28. (canceled)
 29. (canceled)
 30. (canceled)